2-hydroxyethyl acrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

2-hydroxyethyl acrylate is not considered to reproductive toxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 2019 - July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: PAU 0118813

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator (KS)

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 13-14 weeks (male) / about 13 weeks (female)
- Weight at study initiation: mean males= 374 g, mean females=212 g
- Fasting period before study: no
- Housing: Polysulfonate cages: - During pretreatment: up to 5 animals per sex and cage; - During premating: 2 animals per sex and cage; - During mating and postmating: 2 animals per cage (males only) Polycarbonate cages: 1 animal ;
Exceptions: - During overnight mating: 1 male/1 female per cage; - During rearing up to PND 13: 1 dam with her litter
- Diet: ad libitum (Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum (drinking water)
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with ultrapure water and intensely mixed with a homogenizer.During administration, the preparations were kept homogeneous with a magnetic stirrer.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Pregnant animals and their litters were housed together until PND 13. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All measured values for the test item were in the expected range of the target concentrations (90 - 110%) demonstrating the correctness of the preparations. The stability of test substance in drinking water was demonstrated for a period of 7 days at room temperature.

Duration of treatment / exposure:

The duration of treatment covered a 5 weeks in-life period (males) including 14 days mating (mating pairs were from the same test group) as well as a 2-weeks premating period (females), 14 days mating period, 9 days postmating period in one female (for no evidence of sperm), the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.

Frequency of treatment:

once daily

Dose / conc.:

12 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

120 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration

DETAILED CLINICAL OBSERVATIONS: Yes
- The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
Abnormal behavior in “handling”, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos (Protruding eyeball), Assessment of the feces excreted during the examination (appearance/consistency), Assessment of the urine excreted during the examination, Pupil size

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results.
During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
Females with litter were weighed on the day of parturition (PND 0) and on PND 1, 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly. These body weight data were solely used for the calculations of the dose volume; therefore these values are not reported in the Summary but in the Individual Tables.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Generally, food consumption was determined once a week (over a period of 7 days) for male and female parental animals, with the following exceptions:
Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (female parental animals).
Food consumption of the females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
Food consumption of the females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Generally, water consumption was determined once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (female parental animals)
Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
Water consumption of the females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7 and 12-13.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

NEUROBEHAVIOURAL EXAMINATION: Yes
- A functional observational battery (FOB) was performed in the first 5 male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence.
The already single-housed female animals were transferred to new cages before the test, nor was drinking water withdrawn. At least 30 minutes before the start of the FOB the male animals were transferred from group housing to single animal polycarbonate cages type III, for the duration of the test. Drinking water was provided ad libitum, but no food was offered during the measurements. Likewise, food was withdrawn from the female animals for the duration of the test.
The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined: Behavior on removal from cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypy, Gait, Activity/arousal level, Feces (appearance/ consistency) within 2 minutes, Urine (amount/color) within 2 minutes, Rearing within 2 minutes, Other findings

Sensory motor tests/Reflexes
The animals were removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Other findings, Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test

Motor activity measurement
The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

Oestrous cyclicity (parental animals):

For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization (the estrous cycle data of these individuals were not reported and can be found in the raw data). For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Sperm parameters (parental animals):

Parameters examined in F1male parental generations:
testis weight, epididymis weight, stages of spermatogenesis in the testes and histopathology of interstitial testicular cell structures

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. Pups which died accidentally or had to be sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth (PND 0), and on lactation days 4, 7 and 13.
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.

Pup body weight data:
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Anogenital distance
Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.

Nipple/areola anlagen
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen was counted.

Thyroid Hormones
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH).

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Weight parameters: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight); Epididymides; Ovaries; Prostate (ventral and dorsolateral part together, fixed); Seminal vesicles with coagulating glands (fixed); Testes; Thyroid glands (with parathyroid glands) (fixed); Uterus with cervix
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands (fixed); Brain; Heart; Kidneys; Liver; Spleen; Thymus (fixed)
Organ / Tissue fixation:
The following organs or tissues of all parental animals are fixed in 4% neutral buffered formaldehyde solution or modified Davidson’s solution: All gross lesions; Adrenal glands; Aorta; Bone marrow (femur); Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Esophagus; Extraorbital lacrimal glands; Epididymides (modified Davidson’s solution); Eyes with optic nerve (modified Davidson’s solution); Femur with knee joint; Heart; Ileum; Jejunum (with Peyer’s patches); Kidneys; Larynx; Liver; Lungs; Lymph nodes (axillary and mesenteric); Mammary gland (male and female); Nose (nasal cavity); Ovaries (modified Davidson’s solution); Oviducts; Pancreas; Parathyroid glands; Pharynx; Pituitary gland; Prostate; Rectum; Salivary glands (mandibular and sublingual); Sciatic nerve; Seminal vesicles; Skeletal muscle; Spinal cord (cervical, thoracic and lumbar cord); Spleen; Sternum with marrow; Stomach (forestomach and glandular stomach); Target organs; Testes (modified Davidson’s solution); Thymus; Thyroid glands; Trachea; Urinary bladder; Uterus; Vagina

HISTOPATHOLOGY: Yes
All gross lesions; Adrenal glands; Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Epididymides; Eyes with optic nerve; Heart; Ileum; Jejunum; Kidneys; Liver; Lungs; Lymph nodes (axillary and mesenteric); Ovaries; Oviducts; Prostate; Peyer’s patches; Rectum; Sciatic nerve; Seminal vesicles; Skeletal muscle; Spinal cord (cervical, thoracic, lumbar); Spleen; Sternum with marrow; Stomach (forestomach and glandular stomach); Testes; Thymus; Thyroid glands; Trachea; Urinary bladder; Uterus; Vagina

CLINICAL PATHOLOGY:
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in Inter¬national System (SI) units. The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

Hematology
The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA)
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument, Clotting tests were carried out using a ball coagulometer
Clinical chemistry: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.), gamma-Glutamyltransferase (GGT) (gamma -glutamyl) peptide: aminoacid--glutamyl-transferase; EC 2.3.2.2.), sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total proteine, Albumine, Globulins, Triglycerides, Cholesterol, Bile acids

Thyroid Hormones
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Postmortem examinations (offspring):

SACRIFICE
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations.

The remaining pups were sacrificed under isoflurane with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and processed

Statistics:

Weight of the anesthetized animals and absolute and relative organ weights: KRUSKAL-WALLIS H and WILCOXON test
Clinical pathology parameters: KRUSKAL-WALLIS and WILCOXON-Test
Food consumption (parental animals), water consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index: DUNNETT test (two-sided)
Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided)
Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development: WILCOXON test (one-sided+) with BONFERRONI-HOLM
Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index: WILCOXON test (one-sided-) with BONFERRONI-HOLM
% live male day x, %live female day x: WILCOXON test (two-sided)
Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: KRUSKAL-WALLIS and WILCOXON test (two-sided)
Number of cycles and Cycle Length: KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)

Reproductive indices:

see "Any other information"

Offspring viability indices:

see "Any other information"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All mid-and high-dose males showed salivation during the entire study. Salivation was also observed in most of the high-dose females during the premating, mating and gestation period, in all high-dose females during the lactation period, in one mid-dose female during the premating period and in some mid-dose females during the gestation and lactation period.
The temporary salivation was considered to be test substance-induced. Salivation occurred immediately after dosing (up to 2 hours post dosing) in the mentioned animals during the treatment period. It is likely, that this temporary finding was induced by a local affection of the upper digestive tract.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the test groups (1 - 3; 12, 40 and 120 mg/kg bw/d) during the study.
One mid-dose male (No. 30) showed piloerection on study day 21. One high-dose male (No. 32) had an injury of his hindlimbs (toe) during study days 30 - 33. One mid-dose female (No. 122) had an injury of the anogenital region during premating days 2 – 7.
Two high-dose females (Nos. 132 and 138) did not nurse their pups properly during PND 1- 3 and PND 1 - 2, respectively. As a consequence, several pups of high-dose female No. 132 and the only pup of high-dose female No. 138 died within 3 days after they were born.

Further details / tables see attachment

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of all male and female parental animals and body weight change of all female parental animals in all test substance-treated groups were comparable to the concurrent control during the entire study period. Body weight gain was lower in all treated groups at the beginning of exposure (though statistically significant only in the high-dose parental males during study days 0 – 7), suggesting that the animals needed a few days to get adapted to the bolus gavage of this irritating compound. After this adaptation the mean body weight change was generally comparable to the concurrent control values during the rest of the study. The statistically significantly higher mean body weight change in the mid-dose males during study days 13 - 21 was considered to be spontaneous in nature and not treatment related since it was not related to dose.

Further details / tables see attachment

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the high-dose male animals was statistically significantly above the concurrent control values during study days 7 - 13 and 0 - 13 (about 42% and 21%, respectively). The most likely explanation for this is that the animals spilled food.
Food consumption of the high-dose female animals was below control throughout lactation, the difference became statistically significant during PND 7 - 10 (about 23%). Overall the high-dose females consumed about 14% less food than the control females during lactation. Food consumption of the low- and mid-dose males and females during the entire study, and of the high-dose females during the premating and gestation period was comparable to the concurrent control values.
Further details / tables see attachment

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

Water consumption of all male animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study. Water consumption of the mid-dose females during the entire study, and of the low- and high-dose females during the premating and gestation period was comparable to the concurrent control values. Water consumption of the high-dose and low-dose females was statistically significantly below the concurrent control values during PND 6 - 7 (about 17% and 20%, respectively). As there was no dose-response no association to the treatment was assumed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. At the end of the administration period, in males of test groups 1, 2 and 3 (12, 40 and 120 mg/kg bw/d) relative eosinophil counts were decreased (in test group 2 not statistically significantly). The same was true for significantly decreased mean corpuscular hemoglobin concentration (MCHC) in females of test group 3 (120 mg/kg bw/d). However, the values were within historical control ranges (males, relative eosinophils 1.3-2.7 %; females, MCHC 20.84-22.23 mmol/L). In females of test group 2 (40 mg/kg bw/d) total white blood cell (WBC) counts and absolute neutrophil counts were significantly decreased. In females of test group 1 (12 mg/kg bw/d) absolute neutrophil counts were already significantly lower compared to controls. However, the alterations were not dose dependent. Therefore, all mentioned hematology changes were regarded as incidental and not treatment related.

Further details / tables see attachment

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed. At the end of the administration period, in females of test group 3 (120 mg/kg bw/d) creatinine values were significantly decreased, but the values were within the historical control range (females, creatinine 26.9-35.4 µmol/L). In males of test group 2 (40 mg/kg bw/d) inorganic phosphate levels were significantly increased, but this alteration was not dose dependent. Therefore, both mentioned alterations were regarded as incidental and not treatment related.
In parental males of test groups 2 and 3 (40 and 120 mg/kg bw/d) T4 values were significantly increased. However, the means were within the historical control range (males, T4 44.65-73.22 mmol/L). Corresponding TSH values were not changed and within the historical control range (males, TSH 4.32-9.80 µg/L).

Further details / tables see attachment

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation. Apart from transient salivation in three high-dose males and two mid-dose males, the open field observations did not reveal any test substance-related findings in male and female animals of all test groups. One high-dose male had an injury of hindlimbs during the open field observations, which was unrelated to treatment. There were no test substance-related findings in male and female animals of all test groups.
No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly higher values of landing foot-splay test in males of test group 1 and 2 were considered as spontaneous in nature and not treatment-related, as there was no dose-response. In addition, the measured values were within the historical control range (HCD: 9.3 - 13.8 cm). Because of the hindlimbs injury of high-dose male No. 32 no measurement of grip strength of hindlimb and landing foot-splay test was performed with this animal.
No treatment-related, adverse change of motor activity (summation of all intervals) was observed in the male and female animals of all test substance-treated groups in comparison to the concurrent control values.
The mean number of beam interrupts of the high-dose parental males was statistically significantly above the concurrent control values during interval 1. However, the animals of the high-dose group habituated normally to the test conditions afterwards and all other groups including control were rather at the lower end of the historical control range for this interval (HCD: 989.6 – 1349.4). The high-dose value is within the historical control range, thus, considering the regular habituation, this slightly higher activity level is considered to be in the normal range of rat behavior.

Further details / tables see attachment

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the stomach of male and females of test groups 2 and 3.
Forestomach:
Diffuse squamous hyperplasia characterized by a thickening of the epithelial layer including the Margo plicatus and diffuse hyperkeratosis was noted. The lesion was correlated with the macroscopical findings of a thickened Margo plicatus and thickened wall. Additionally, erosions and ulcerations were present in multiple males and females of the test group 3. The epithelium surrounding the ulcerations and erosions displayed a pronounced squamous hyperplasia and hyperkeratosis. An inflammatory infiltrate, consisting of mainly neutrophils, lymphocytes and plasma cells was seen in the underlying tissue, as well as a submucosal edema. The squamous hyperplasia seen in one control male and one male/female each in test group 1 is regarded as incidental and not-treatment-related. In addition, the erosion/ulceration in one control male is a spontaneous background lesion.
Glandular stomach:
Multifocal edema and hyperemia of the lamina propria, often accompanied by an attenuation of the overlying epithelial layer was noted in male and female animals of test groups 2 and 3 and is assumed as consequence of a local irritating effect of the test substance. Additionally, ulcerations and / or erosions without a dose-dependency were found in the glandular stomach of males and females correlating with macroscopic findings. Since no dose-dependency could be observed and animals affected in test group 1 and 2 did not display the described test substance related histological lesions in the forestomach (diffuse squamous hyperplasia, submucosal edema and erosion/ulceration) and glandular stomach (edema/hyperemia in the lamina propria), this finding is regarded as a not treatment-related spontaneous background lesion.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Further details / tables see attachment

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was equal: 4.0 days in all test groups 0 - 3, respectively.

Further details / tables see attachment

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls.

Further details / tables see attachment

Reproductive performance:

no effects observed

Description (incidence and severity):

For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (0 - 3). Fertility was proven for all F0 parental males within the scheduled mating interval for F1 litter. Thus, the male fertility index was 100% in all test groups. The female mating index calculated after the mating period for F1 litter was 100% in all test groups (0 - 3). All female rats delivered pups or had implants in utero: The fertility index was 100% in all test groups. The mean duration of gestation values varied between 22.0 / 22.0 / 22.0 and 22.4 in test groups 0 - 3, respectively. The gestation index was 100% in all test groups 0 - 3.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (11.4 / 11.2 / 12.8 and 12.4 implants/dam in test groups 0 - 3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups (3.8 / 0.8 / 4.7 and 11.6 mean% in test groups 0 - 3, respectively), and all values were below or within the historical control range of the test facility (0.9 – 16.8 mean%) and the mean number of F1 pups delivered per dam remained unaffected (10.9 / 11.1 / 12.3 and 11.5 pups/dam in test groups 0 - 3, respectively).

Further details / tables see attachment

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects in highest dose observed

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced food consumption, increased liver weights

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

12 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: gastrointestinal pathology

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups

Further details / tables see attachment

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99.1%/ 100% / 97.7% and 83.6% in test groups 0 - 3, respectively. The apparently lower pup survival rate in the high-dose group was caused by the two individual dams 132 and 138. Dam 132 had a large litter (14 pups) and was not able to nurse all her pups properly. Consequentially 5 pups died from undernutrition within 3 days after they were born. Dam 138 had only one pup, which also died from undernutrition 3 days after it was born. The pups surviving index indicating pup survival during lactation (PND 4 - 13) was 100% in all test groups.
Further details / tables see attachment

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.

Further details / tables see attachment

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In male and female pups at PND13 (test groups 11, 12 and 13; 12, 40 and 120 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Further details / tables see attachment

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

In general, the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature. The statistically significantly shifted in sex ratio in the high-dose group on PND 0 (males 62.8*[*p<=0.05] versus [vs.] 43.8 in control and females 37.2* vs. 56.2 in control) was slightly outside the historical control range (HCD: 36.6% - 60.5% (males), 41.5% - 63.4% (females)). Nevertheless, it was assessed as not treatment-related, because pup necropsy at PND 13 revealed no morphological evidence for virilization and none of the other endocrine-sensitive parameters (e.g. pup weight, AGD, nipple retention) gave any indication of a test substance-related effect in any of the dose levels (see below). Thus, these values were most likely outliers and considered to be spontaneous in nature and not treatment related.

Further details / tables see attachment

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.

Further details / tables see attachment

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Further details / tables see attachment

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few individual pups showed spontaneous findings at gross necropsy, such as discolored testis, empty stomach, dilated renal pelvis, post mortem autolysis, partly cannibalized, absent mouth, absent jaw and dilated ureter. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

Further details / tables see attachment

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects at highest dose

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

The results in tabular form are provided in the attached background material as pdf. Results for all organ weights (absolute and relative) are provided as separate pdf.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - with developmental immunotoxicity (Cohorts 1A, 1B without extension, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

According to ECHA Compliance Check Decision number CCH-D-2114382275-45-01/F
- Premating exposure duration for parental (P0) animals : Ten weeks premating exposure duration for the parental (P0) generation, according to ECHA Compliance Check Decision.
- Basis for dose level selection : based on the results of the OECD 422 (2018) (Dose level setting shall aim to induce systemic toxicity at the highest dose level, according to ECHA Compliance Check Decision)
- Exclusion of extension of Cohort 1B, according to ECHA Compliance Check Desision
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B , according to ECHA Compliance Check Decision
- Inclusion of developmental immunotoxicity Cohort 3, according to ECHA Compliance Check Decision
- Route of administration : oral (gavage), according to ECHA Compliance Check Decision
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: The rat is the preferred animal species for reproduction studies according to test guidelines. This strain was selected since extensive historical control data were available for Wistar rats.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany, Sulzfeld,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 weeks
- Weight at study initiation: weight variation did not exceed 20 percent of the mean weight of each sex.
- Housing: During the study period, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany with the following exceptions:During overnight mating (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually in Polycarbonate cages type III (supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany).
Dams and their litters were housed together until PND 21/22 in Polycarbonate cages type III.
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland (ad libitum)
- Water (e.g. ad libitum): drinking water (ad libitum)
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and intensely mixed with a magnetic stirrer until it was completely dissolved. During administration, the preparations were kept homogeneous with a magnetic stirrer.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days
- Proof of pregnancy:sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in drinking water over a period of 7 days at room temperature has been verified prior to the start of the study in a similar batch. At the beginning of administration, towards the middle and towards the end of administration each 1 sample was taken from the low, mid and high concentration for a concentration control analysis.

Duration of treatment / exposure:

F0 males: 10 weeks (premating) + 2 weeks (mating) + max. 6 weeks (post-mating)
F0 females: 10 weeks (premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 animals (Cohort 1A and 1B): post weaning until an approx. age of 12-13 weeks
F1 animals (Cohort 3): post weaning until an approx. age of 8-9 weeks

Frequency of treatment:

once daily
Females in labor were not treated.

Details on study schedule:

F0 generation animals and their progeny:
The male and female rats were about 4 weeks old when they arrived from the breeder. During an acclimatization period of about 6 days, animals with lowest and highest body weights were eliminated from the study and used for other purposes. The 100 male and 100 female animals required for the study were about 5 weeks old at the beginning of treatment and their weight variation did not exceed 20 percent of the mean weight of each sex. The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight one day before the beginning of the administration period (day -1). After the acclimatization period, the test substance was administered to the animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (drinking water), in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight. After a minimum of 10 weeks after the beginning of treatment, males and females from the same dose group were mated, overnight at a ratio of 1 : 1 (for details see: Pairing of F0 generation parental animals). The females were allowed to deliver and rear their pups (F1 generation pups) until PND 4 (standardization) or PND 21 or 22 (depending on the cohort). Pups of the F1 litter were selected (F1 rearing animals) and assigned to 3 different cohorts which were subjected to specific postweaning examinations. On PND 4 blood samples were collected from 10 surplus (culled) F1 pups per sex and group. On PND 22 blood samples were collected from 10 surplus F1 pups per sex and group. Urine samples were taken from 10 selected F0 parents and all cohort 1A animals shortly before sacrifice. Blood samples were taken from 10 animals per test group of the F0 parental animals and cohort 1A animals. Before weaning of the F1 pups the F0 generation parental male animals were sacrificed. After weaning of F1 pups the F0 generation parental female animals were sacrificed.

F1 rearing animals:
Before weaning of the F1 generation pups on PND 21, 55 male and 55 females per group were randomly selected (selection see below), to be placed into cohorts according to the scheme. Obvious runts (those pups whose body weight was greater than 25% below the mean body weight of the control group, separate for sexes) were not included.
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)
Cohort 3: One male or one female/litter (10/sex/group)
Selected F1 offspring received the test substance daily by gavage until one day before sacrifice. In addition, 10 male and 10 female pups were randomly selected from the control group to build test group 14 (positive control group).

Standardization of litters (culling) of F1 generation:
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 5 male and 5 female pups (always the first 5 pups/sex and litter were taken for further rearing). If individual litters did not have 5 pups/sex, the litters were processed in such a way that the most evenly distributed 10 pups per litter were present for further rearing (e.g., 6 male and 4 female pups). Surplus animals were sacrificed according to 3.8.2.7. Standardization of litters was not performed in litters with <= 10 pups.

Pups after standardization/weaning:
With the exception of those F1 generation pups, which were chosen as F1 rearing animals, and those F1 pups, which were chosen for blood sampling on PND 4 and 22, all pups were sacrificed under isoflurane anesthesia with CO2 after standardization or weaning. The pups chosen for blood sampling were sacrificed by decapitation under isoflurane anesthesia. All culled pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and visceral) examination. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a caseby-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

F0: 25 animals per sex per dose
F1 cohort 1A: 20 animals per sex per dose
F1 cohort 1B: 25 animals per sex per dose
F1 cohort 3: 10 animals per sex per doese
F1 positive control for immuotox: 10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the results of the OECD 422:
15 mg/kg body weight/day as low dose level
50 mg/kg body weight/day as mid dose level
150 mg/kg body weight/day as high dose level

Positive control:

Cyclophosphamide monohydrate (control group for immunotoxiciy):
The positive control substance solution in drinking water was prepared before the beginning of the administration period. For the preparation of the administration solution the positive control substance was weighed in a weighing boat depending on the dose group and transferred quantitatively in a graduated flask, topped up with drinking water and subsequently thoroughly mixed by a magnetic stirrer until it was completely dissolved. Thereafter the positive control preparation was split in “Nalgene Dosen” (40 ml each) and frozen by -18°C.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were performed in all F0 parental animals (once before the beginning of the administration period on day 0) and F1 animals in cohorts 1A, 1B and 3 at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed: Abnormal behavior in handling; Fur; Skin; Posture; Salivation; Respiration; Activity/arousal level; Tremors; Convulsions; Abnormal movements; Gait abnormalities; Lacrimation; Palpebral closure; Exophthalmos (Protruding eyeball); Assessment of the feces excreted during the examination (appearance/consistency); Assessment of the urine excreted during the examination; Pupil size

BODY WEIGHT: Yes / No / No data
In general, the body weight of the male and female F0 parental animals and F1 rearing animals was determined once a week at the same time of the day (in the morning). The body weight of the F1 rearing animals was determined on the first day of test substance administration and then once a week at the same time of the day (in the morning), with the following exceptions:
• During the mating period of the F0 parental animals, the females were weighed on the day of positive evidence of sperm GD 0 and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition PND 0 and on PND 1, 4, 7, 10,14, 18 and 21.
The body weight change of the animals was calculated from these results. Females without positive evidence of sperm, females without litter and females after weaning (PND 21/22), were weighed once a week together with the males. These body weight data were solely used for the calculations of the dose volume; therefore these values are not reported in the Summary but in the Individual Tables.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Generally, food consumption was determined once a week (over a period of 7 days) for male and female F0 parental animals and F1 rearing animals, with the following exceptions:
• Food consumption was not determined after the 10th premating week (male F0 animals).
• During pregnancy, food consumption of the F0 females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• During lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Generally, water consumption was determined once a week (over a period of 3 or 4 days) for male and female F0 parental animals and F1 rearing animals (except of the positive control animals, test group 14), with the following exceptions:
• Water consumption was not determined after the 10th premating week (male F0 animals) • During pregnancy, water consumption of the F0 females with evidence of sperm was determined for GD 0-1, 3-4, 7-8, 10-11, 14-15, 17-18 and 19-20.
• During lactation, water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period, in the females after weaning.

CLIINICAL PATHOLOGY in F0 parental animals:
Samples were withdrawn from 10 F0 parental males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a random- ized sequence. The list of randomization instructions was compiled with a computer. In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instruc- tions was compiled with a computer). The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units.
Hematology:
The following parameters were determined :
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)
- Prothrombin time (Hepato Quick’s test; HQT)
Clinical chemistry:
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
Hormones:
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisas was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
Urinalysis:
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
-Cholesterol (CHO

Oestrous cyclicity (parental animals):

Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female and cohort 1A and 1B female with scheduled sacrifice.

Sperm parameters (parental animals):

After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and all cohort 1A males sacrificed on schedule:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
Pup number and status at delivery:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.
Pup viability/mortality:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in section “Pup necropsy observations”. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21.
Sex ratio:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.
Pup clinical observations:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.
Pup body weight data:
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7, 14 and 21. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters. In the summary tables pup body weights and pup body weight gains are listed for males, females and males + females. Additionally, the body weight of all F1 rearing animals was determined on the day of vaginal opening and preputial separation.
Anogenital distance:
Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.
Nipple/areola anlagen:
All surviving F1 male pups were examined for the presence of nipple/areola anlagen on PND 13 and were re-examined on PND 20. The number of nipple/areola anlagen will be counted.
Vaginal opening:
All female F1 pups selected to become the F1 rearing animals (cohort 1A, 1B and 3) were evaluated daily for vaginal patency beginning on PND 27. On the day of vaginal opening the body weights of the respective animals were determined.
Preputial separation:
All male F1 pups selected to become the F1 rearing animals (cohort 1A, 1B and 3) were evaluated daily for preputial separation beginning on PND 38. On the day of preputial separation the body weights of the respective animals were determined.

CLINICAL PATHOLOGY in cohort 1A animals:
Samples were withdrawn from 10 cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a random- ized sequence. The list of randomization instructions was compiled with a computer.
In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instruc- tions was compiled with a computer). The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units.
Hematology:
The following parameters were determined :
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)
- Prothrombin time (Hepato Quick’s test; HQT)
Clinical chemistry:
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
Hormones:
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisas was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
Hormones in PND 4 and 22 F1-offspring:
Blood sampling
Blood samples were withdrawn from 10 surplus (culled) PND 4 offspring (as far as possible of different litters) per sex and group. PND 4 samples were pooled per sex and litter if the available amount is not sufficient for a hormone analysis. Blood samples were withdrawn from 10 surplus PND 22 offspring (as far as possible of different litters) per sex and group. The blood samples was collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisas was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
Urinalysis:
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
-Cholesterol (CHO

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: yes
Developmental immunotoxicity examinations in cohort 3 animals and positive control animals
Cyclophosphamide dependent immune system response:
Ten male and ten female offspring derived from test group 00 (as far as possible from different litters) were selected at weaning to become a positive control group in this study. These animals were treated with Cyclophosphamide monohydrate to prove the functional responsiveness of major components of the immune system of the rats against an immunosuppressant. The animals were treated by daily oral gavage from PND 35 onwards, for about four weeks. The following dose level of Cyclophosphamide monohydrate was selected to be sufficient to cause immunosuppressive activity as positive control substance: 4.5 mg/kg body weight/day: as dose level with expected immunosuppressive effects The oral route was selected since oral administration of Cyclophosphamide effectively causes the desired immunosuppressive effect.
POSITIVE CONTROL SUBSTANCE PREPARATIONS AND ADMINISTRATION:
Oral administration by gavage using 3 or 5 mL syringes, once daily. 10 mL/kg body weight; the body weight determined most recently were used to calculate the administration volume. Duration ofadministration about 4 weeks. Six days after immunization blood samples were taken by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer.

T-cell dependent antibody response:
All males and females of cohort 3 and the positive control animals were used to assess the functional responsiveness of major components of the immune system to a T-cell dependent antigen, sheep red blood cells (SRBC). For this purpose, the Anti SRBC-IgM ELISA of Life Diagnostics Inc, West Chester, USA (cat. no. 4200-2), was performed. Each sample was di- luted 1:500. SRBC-IgM concentrations outside the standard curve range were measured in a second test run with an appropriate dilution. Generally, two in-house controls were measured with each test run. The ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maenne- dorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Splenic lymphocyte subpopulation analysis:
The immunophenotyping was performed with a FACSLyric flow cytometer (Becton Dickinson, Heidelberg, Germany). 10 males and 10 females of cohort 1A were used to perform a splenic lymphocyte subpopulation analysis using one half of the spleen.
Parameter: B lymphocytes (B_SPL); T lymphocytes (T_SPL), CD4+ lymphocytes (CD4_SPL); CD8+ lymphocytes (CD8_SPL); Natural killer cells (NK_SPL)

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE / NECROPSY
All F0 generation parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.) The female animal No. 139 (F0 generation, parental animals, test group 02) died intercurrently they were necropsied and assessed by gross pathology as soon as possible after their death.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Caudae epididymides
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

Organ/tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson ´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens
The ovaries and eyes with optic nerve of animals that died or were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution. The left testis and left epididymis of all male F0 parental animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.
For technical reasons, after about 24 hours fixation the ovaries of all F0 females of all test groups were transferred to 70% ethanol.
The uteri of all cohabited female F0 generation parental animals were examined for the pres- ence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.

Peer review:
After completion of the histopathological assessment by the study pathologist an internal peer review was performed by Dr. Maria Cecilia Rey Moreno (BASF SE, Ludwigshafen) including forestomach and glandular stomach of all examined male and female animals of the F0 parental generation and of the F1 cohort 1A rearing animals. Results presented in the pathology report reflect the consensus opinion of the study pathologist and the peer review pathologist.

Postmortem examinations (offspring):

SACRIFICE / NECROPSY
All animals of F1 generation, rearing animals, cohort 1A were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were nec- ropsied and assessed by gross pathology; special attention being given to the reproductive organs.) The female animal No. 374 (F1 generation, rearing animals, cohort 1A, test group 13) was sacrificed moribund, they were necropsied and assessed by gross pathology as soon as possible after their death.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Caudae epididymides
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

Organ/tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson ´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens
The ovaries and eyes with optic nerve of animals that died or were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution. The left testis and left epididymis of all Cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.
For technical reasons, after about 24 hours fixation the ovaries of all cohort 1A females of all test groups were transferred to 70% ethanol.
Spleens of 10 animals per sex per group of cohort 1A were split in two comparable parts (transversally). One part of the spleen was fixed in 4% neutral buffered formaldehyde and afterwards embedded in paraplast. The other part of the spleen was frozen at -80o C, being used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells).

Differential Ovarian Follicle Count (DOFC) in cohort 1A females:
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al. (1993). In general, sections were prepared with 2 - 3 μm thickness and serial sections were taken every 100 μm to complete about 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, H&E-stained slides were prepared from all cut levels. Counting was performed on slides digitalized with a Hamamatsu NanoZoomer 2.0 slide scanner using the Hamamatsu viewing software (NDP.view).
Peer review:
After completion of the histopathological assessment by the study pathologist an internal peer review was performed by Dr. Maria Cecilia Rey Moreno (BASF SE, Ludwigshafen) including forestomach and glandular stomach of all examined male and female animals of the F0 parental generation and of the F1 cohort 1A rearing animals. Results presented in the pathology report reflect the consensus opinion of the study pathologist and the peer review pathologist.

NECROPSY Cohort 1B:
All cohort 1B animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. Animals which died intercurrently (male animals No. 475 (test group 12) and No. 487 (test group 13) were necropsied as soon as possible after their death and assessed by gross pathology.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Cauda epididymis
4. Epididymides
5. Liver
6. Ovaries
7. Pituitary gland (fixed)
8. Prostate (ventral and dorsolateral part together, fixed)
9. Testes
10. Seminal vesicles including coagulating gland (fixed)
11. Uterus (with cervix)
All paired organs were weighed together (left and right)

Organ/Tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Cervix uteri
4. Coagulating glands
5. Epididymides (fixed in modified Davidson ´s solution)
6. Liver
7. Ovaries (fixed in modified Davidson´s solution)
8. Pituitary gland
9. Prostate
10. Seminal vesicles including coagulating glands
11. Stomach (forestomach and glandular stomach)
12. Testes (fixed in modified Davidson ´s solution)
13. Uterus
14. Vagina
The testes and epididymides of the animals that died (Nos. 475, test group 12 and 487, test group 13) were fixed in 4% buffered formaldehyde solution. For technical reasons, after about 24 hours fixation the ovaries of all cohort 1B females of all test groups were transferred to 70% ethanol.

Histotechnical processing and examination by light microscopy was not performed. For technical reasons, the ovaries of all cohort 1B females of all test groups were embedded in paraplast.

NECROPSY Cohort 3 and positive control:
All Cohort 3 animals and the animals of the positive control were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.
HISTOPATHOLOGY / ORGAN WEIGHTS:
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Spleen
3. Thymus

Organ/Tissue fixation.
The following organs or tissues were fixed in 4% buffered formaldehyde solution:
1. All gross lesions
2. Spleen
3. Thymus

Histotechnical processing and examination were not performed.

NECROPSY surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts).

All surplus F1 generation pups that were not used for the following organ weight determinations were sacrificed under isoflurane anesthesia with CO2. The selected pups for organ weight determination were sacrificed by decapitation under isoflurane anesthesia. All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.
HISTOPATHOLOGY / ORGAN WEIGHTS:
The following weights were determined in up to 10 animals per sex per group sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Spleen
4. Thymus (fixed)

Organ/Tissue fixation.
The following organs or tissues of up to 10 animals per sex per group were fixed in 4% neutralbuffered formaldehyde solution:
1. All gross lesions
2. Brain
3. Mammary gland (male and female)
4. Spleen
5. Thymus
6. Thyroid glands

Histotechnical processing and examination was not performed.

Statistics:

Water consumption (parental and rearing animals), food consumption (parental and rearing animals), body weight and body weight change (parental animals, rearing animals and pups; for the pup weights, the litter means were used), gestation days, duration of sexual maturation (days to vaginal opening, days to
preputial separation), anogenital distance, anogenital index: DUNNETT test (twosided)
Male and female mating indices, male and female fertility indices, gestation index, females mated, females pregnant, females delivering, females with liveborn
pups, females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided)
Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal Loss, nipple development: WILCOXON test (one-sided+)
Implantation sites, pups delivered, pups liveborn, live pups day x, viability index, lactation index: WILCOXON test (one-sided-)
% live male day x, % live female day x: WILCOXON test (twosided)
Number of cycles and Cycle Length: KRUSKAL-WALLIS test (two-sided), WILCOXON-test (twosided)
Blood parameters and splenic lymphocytes subpopulations: Non-parametric one-way analysis using KRUSKAL-WALLIS test., WILCOXON-test (two-sided), WILCOXON-test (one-sided)
Urinalysis parameters (apartfrom pH, urine volume, specific gravity, color and turbidity: WILCOXON-test (one-sided)
Urine pH, volume and specific gravity: Non-parametric one-way analysis using KRUSKAL-WALLIS test, WILCOXON-test (two-sided)
Urine color and turbidity: Urine color and turbidity are not evaluated statistically.
Spermanalysis parameters:WILCOXON-test (one-sided)
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided), WILCOXON-test (two-sided), WILCOXON-test (two-sided) f
DOFC (differential ovarian follicular count): WILCOXON-test (one-sided-)

Reproductive indices:

Male mating index (%) = number of males with confirmed mating / number of males placed with females x 100
Male fertility index (%) = number of males proving their fertility / number of males placed with females x 100
Female mating index (%) = number of females mated / number of females placed with males x 100
Female fertility index (%) = number of females pregnant / number of females mated x 100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100

Offspring viability indices:

Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100
Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations x 100
Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth x 100
Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All high-dose and nearly all mid-dose males showed salivation at least once during the study. Likewise, salivation was observed in all high-dose females as well as about half of the middose females at least once during the study (including premating, mating, gestation and lactation periods). In all these cases salivation appeared in a short time period immediately after administration and disappeared within minutes or a maximum of two hours after administration. This temporary salivation was considered to be test substance-induced. No salivation was noted in the low-dose group.

Clinical observations for males and females (except gestation and lactation period):
One mid-dose male animal (No. 60) showed eye discharge (left, red) during study days 20 - 114. One control female animal (No. 119) had a protruding eyeball (right) during premating days 20 – 75 and during the mating period (days 1 - 3). One low- and one mid-dose female (Nos. 130 and 154, respectively) showed reduced nutritional condition (grade: slight to severe with indrawn flanks and grade: slight to moderate with indrawn flanks, respectively) during premating days 47 - 70 and 52 - 75, respectively. These observations were considered not to be associated with the test compound.

Clinical observations for females during gestation of F1 litters:
One control female animal (No. 119) had a protruding eyeball (right) during the entire gestation period. One low-dose female animal (No. 146) had vaginal discharge (reddish) on GD 18. One mid-dose female (No. 154) had piloerection, reduced feces and reduced nutritional condition during GD 8 - 57.
One sperm positive low-dose female (No. 130) and one sperm positive mid-dose female (No. 154) did not deliver F1 pups and had no implants in the uterus. These observations were considered not to be associated with the test compound.

Clinical observations for females and offspring during lactation of F1 litters: One control female animal (No. 119) had a protruding eyeball (right) during lactation period.

Detailed clinical observations (DCO):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
One mid-dose male (No. 60) showed eye discharge (left, red) during DCO days 22 - 106. One control female (No. 119) had a protruding eyeball (right) during DCO days 22 - 127. One lowdose female (No. 130) had reduced nutritional conditions (grade: slight, with indrawn flanks) during DCO days 50 - 64. One low-dose female (No. 146) had vaginal discharge (reddish) on DCO day 99. One mid-dose female (No 154) showed reduced nutritional condition (grade: slight to moderate, with indrawn flanks) during DCO days 57 - 71 and 120 - 127 and piloerection during DCO days 120 - 127. These observations were considered not to be associated with the test compound.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test substance-related mortalities in any of the groups.
One low-dose female animal (No. 139) showed hypothermia, piloerection, and blood in bedding on GD 23, had semiclosed eyelids (both), all pups stillborn and pups still palpable in abdomen after delivery on PND 0, hypothermia, piloerection, pale skin (entire body) and hunched posture during PND 0 - 1, smeared fur (anogenital region, light yellow) on PND 1 and was found dead on PND 2. It showed an adenocarcinoma in the uterus and multifocal necrosis in the liver, which were unrelated to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were statistically significant increases or decreases in body weight/body weight change in F0 animals of various dose groups, which were small, temporary, not dose-related and rather inconsistent and thus considered to be of no toxicological relevance:
• decreased body weight change in high-dose males on study days 21 - 49, 84 - 105 and 0 – 112
• decreased body weight change in mid-dose males on study days 21 - 28, 35 - 42, 49 - 56 and 0 - 112
• increased body weight change in low-dose males on study days 70 - 77
• decreased body weight change in mid-dose females on premating days 14 - 21 and 0 - 70
• decreased body weights in mid-dose males on study days 84 - 91 and on day 105
• decreased body weights in mid-dose females on premating days 28 - 35, 49 - 70 and on PND 1 and 14

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of all male and female animals of all test substance-treated groups was generally comparable to the concurrent control values throughout the entire study. There were statistically significant increases or decreases in food consumption in F0 animals of various dose groups, which were small, temporary and rather inconsistent and thus considered to be spontaneous in nature and not treatment-related:
• increase in high-dose males on study days 0 – 21
• increase in high-dose females on premating days 35 - 42 and 0 - 70
• decrease in low-dose males on study days 42 – 49
• decrease and in low-dose females on PND 4 – 7.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

Water consumption of all male and female animals of all test substance-treated groups was generally comparable to the concurrent control values throughout the entire study. The statistically significantly increased water consumption of the high-dose females during premating days 0 - 3 (about 11%) was most likely spontaneous in nature and not treatmentrelated

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period, in F0 males of test group 03 (150 mg/kg bw/d) absolute and relative neutrophil cell counts were significantly increased whereas relative lymphocyte counts were significantly decreased. In parental females of test group 03 (150 mg/kg bw/d red blood cell (RBC) counts, hemoglobin and hematocrit values were significantly decreased whereas absolute reticulocyte counts were significantly increased. These changes were regarded as treatment related and adverse.
In parental males of test group 03 (150 mg/kg bw/d) absolute reticulocyte counts were significantly increased whereas relative basophil counts were significantly decreased, but the values were within historical control ranges (males, absolute reticulocytes 105.7-175.0 Giga/L, relative basophils 0.1-0.4 %). In males of test group 02 (50 mg/kg bw/d) hematocrit values were significantly higher compared to controls, but the alteration was not dose dependent. Therefore, these changes were regarded as incidental and not treatment related.
In F0 females, absolute lymphocyte counts were significantly increased, but the values were within the historical control range (females, absolute lymphocytes 1.93-3.07 Giga/L). Therefore, this change was regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period, in males of test group 03 (150 mg/kg bw/d) cholesterol values were significantly increased and they were above the historical control range (males, cholesterol 1.56-2.10 mmol/L). However, this is an isolated liver parameter change. Therefore, it was regarded as maybe treatment related, but non-adverse. In females of this test group urea and inorganic phosphate values were significantly increased whereas globulin values were significantly decreased. Inorganic phosphate levels were already significantly higher in females of test group 02 (50 mg/kg bw/d). However, all values among these females were within historical control ranges (females, urea 5.63-8.76 mmol/L, inorganic phosphate 0.98- 1.57 mmol/L, globulins 24.80-27.79 g/L). therefore, the mentioned changes were regarded as incidental and not treatment related.
In males of test group 02 (50 mg/kg bw/d) glucose values were significantly decreased and in males of the same test group calcium values were significantly decreased. However, the changes were not dose dependent, and therefore, they were regarded as incidental and not treatment related.

Endocrine findings:

no effects observed

Description (incidence and severity):

Neither the anogenital distance/index nor the check for the presence of nipples/areolas, both very sensitive marker of potential endocrine-mediated imbalances, revealed any test substance-related effects.
A statistically significant delay in vaginal opening of about one day beyond the concurrent control was observed in the female F1 offspring of the high-dose group (150 mg/kg bw/d). The delay is, however, within the historical control range of the test facility. This apparent delay was mainly due to 1 high-dose individual entering puberty after the age of 43 days. This and the still rather small average difference to the control of about one day indicates that the later onset
of puberty in the high-dose group is not a specific effect on the timing of puberty. In addition, none of the other endocrine-sensitive parameters like anogenital distance, or estrous cyclicity in the F1A offspring brought beyond puberty, or the integrity of sexual organs in these females including differential ovarian follicle count, indicated any effect of the test item. Thus, the apparently later entry of high-dose offspring into female puberty is likely to be incidental.
Entry into male puberty was not influenced by the test substance as indicated by an unchanged timing of preputial separation.
Measurement of thyroid hormones revealed no effect caused by the test substance, neither in the F0 parental animals nor in the F1 offspring.
At the end of the administration period in F0 males of test groups 01, 02 and 03 (15, 50 and 150 mg/kg bw/d) T4 values were significantly higher compared to controls. The mean values of all three test groups were above the historical control range (males, T4 44.65-73.22 nmol/L). However, TSH values were not changed among these individuals. No histologic finding in the thyroids was observed although relative thyroid weight in males of test groups 02 and 03 were significantly increased, but they were within the historical control range. Therefore, the isolated T4 increase in F0 males of test groups 01, 02 and 03 was regarded as maybe treatment related, but non-adverse (ECETOC Technical Report No 85, 2002).
In parental females of test group 03 (150 mg/kg bw/d) T4 values were significantly increased. The mean value was above the historical control range (females, T4 24.28-43.26 nmol/L). However, TSH values were not significantly changed and no histologic finding in the thyroids was observed. Therefore, the isolated T4 increase in F0 females of test group 03 was regarded as maybe treatment related, but non-adverse (ECETOC Technical Report No 85, 2002).

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the fore- and glandular stomach in male and female animals of test groups 02 and 03.
Secondary findings were noted in the pancreatic lymph node in 10/25 male animals of test group 03. This lymph node is not a protocol organ and was only collected in the animals where it was macroscopically enlarged.

Forestomach:
In the forestomach, erosion/ulcer were noted in many animals of both sexes of test group 03, correlating to the gross finding “focus”. The forestomach also showed a thickening of the squamous cell layer, most frequently affecting the region of the margo plicatus, which was recorded as squamous hyperplasia, margo plicatus, diffuse. This was seen in both test group 02 and with increased incidence and severity in test group 03 animals of both sexes. This finding correlated to the gross finding “margo plicatus thickened”. In a few cases, the thickening of the squamous layer extended throughout the whole section of the forestomach, this was recorded as hyperplasia squamous cell diffuse. This was only seen in few animals of both sexes of test group 03.

Glandular stomach:
Multifocal degeneration/regeneration was seen in many animals of both sexes in test group 03. It was characterized by a loss of the normal architecture of the glandular mucosa, which was replaced by more basophilic undifferentiated cells. This finding was especially prominent adjacent to the margo plicatus. Erosion/ulcer were seen with increased incidence in treated animals of both sexes.

Pancreatic lymph node:
The gross finding “enlarged” in test group 03 correlated with increased cellularity of plasma cells and lymphocytes and lymphoid cysts and in a few cases with sinus histiocytosis. This was assumed to be a reaction to the ulcers and erosions seen in the fore- and glandular stomach. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility:
The female animals (No.111 (test group 00), 130 (test group 01), and 199 (test group 03)), which were not pregnant as well as their male mating partners (No.11 (test group 00), 30 (test group 01), 54 (test group 02), and 99 (test group 03)) did not show relevant histopathological findings consistent with impaired fertility. Female animal No 154 showed an atrophic uterus, cervix, and vagina which was assumed to have contributed to the impaired fertility of this mating pair.

Decedents:
The female animal No. 139 of test group 01 died spontaneously. It showed an adenocarcinoma in the uterus and multifocal necrosis in the liver, which was unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups 01-03. The mean estrous cycle duration was comparable: 4.0 / 4.1 / 4.1 and 4.1 days in test groups 01-03, respectively.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermanalysis:
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatmentrelated effects were observed.

Reproductive performance:

no effects observed

Description (incidence and severity):

For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (00 - 03). Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One low-dose male (No. 30) and one mid-dose male (No. 54) did not generate F1 pups. Thus, the male fertility index ranged between 96% and 100% without showing a doseresponse. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until copulation was detected (GD 0) varied between 2.4 and 2.9 days without any relation to dosing. All female rats delivered pups or had implants in utero with the following exceptions:
• Test group 01: female No. 130 (mated with male No. 30) did not become pregnant
• Test group 02: female No. 154 (mated with male No. 54) did not become pregnant
Except of female animal No 154, which had an atrophic uterus, cervix, and vagina, the apparently infertile female rats did not show histopathological findings that could explain infertility.
The fertility index ranged between 96% and 100% without showing any relation to dosing. The mean duration of gestation was comparable in all test groups (i.e. between 22.2 and 22.4 days).
The gestation index was 96% / 95.8% / 100% and 96% in test groups 00 - 03.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (11.9 / 11.9 / 11.8 and 12.1 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (11.2 / 11.2 / 5.4 and 10.8 mean% in test groups 00 - 03, respectively)., and the mean number of F1 pups delivered per dam remained unaffected (11.4/ 10.6 / 11.1 and 11.2 pups/dam in test groups 00 - 03, respectively).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 97.4% / 100% / 97.5% and 98.2% in test groups 00 - 03, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups. The rate of liveborn pups was not affected by the test substance, as indicated by live birth index of 99.3% / 94.5% / 98.9% and 97.4% in test groups 00 - 03. Moreover, the number of stillborn pups was comparable between the groups. Thus, the test substance Hydroxypropyl acrylate did not adversely affect reproduction and delivery of the F0 generation parental females.

Dose descriptor:

NOAEL

Remarks:

fertility/reproductive performance

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A:
All high- and most of the mid-dose males and females showed salivation at least once during the study. In all these cases salivation appeared in a short time period immediately after administration and disappeared within minutes or a maximum of two hours after administration. This temporary salivation was considered to be test substance-induced. No salivation was noted in the low-dose group.
One mid-dose male animal (No. 246) had an encrusted eye (right, reddish) during study days 0 - 64, one mid-dose male animals (No. 248) had a protruding eyeball (right) during study days 21 - 64 and one high-dose male animal (No. 280) had a protruding eyeball (left) during study days 20 - 64. These observations were considered not to be associated with the test compound.
Detailed clinical observations (DCO):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups. One female animal (No. 374) of test group 13 was sacrificed moribund prior to DCO day 0. One mid-dose male animal (No. 246) had an encrusted eye (right, reddish) during DCO days 0 - 56, one mid-dose male animals (No. 248) had a protruding eyeball (right) during DCO days 21 - 56 and one high-dose male animal (No. 280) had a protruding eyeball (left) during DCO days 21 – 56. None of these findings was considered to be associated with the treatment.

F1 generation parental animals, Cohort 1B:
Nearly all high-and most of the mid-dose males and females showed salivation at least once during the study. In all these cases salivation appeared in a short time period immediately after administration and disappeared within minutes or a maximum of two hours after administration. This temporary salivation was considered to be test substance-induced. No salivation was noted in the low-dose group.
One mid-dose male animal (No. 451) had a semiclosed eyelid (right) on study day 0, two middose male animals (No. 457 and 473) had encrusted eyes (both, reddish) during study days 0- 56 and 0 - 59, respectively. One high-dose male animal (No. 498) had a protruding eyeball (right) during study days 0 – 7, a protruding eyeball (right, dark red) during study days 7 – 15, a protruding eyeball (right, light red) on study days 16, a small eye (right) on study day 17, a discolored eye (right, light red) on study day 17 and an injury (right, cornea bleb) during study days 17 - 59. These observations were considered not to be associated with the test compound.
Detailed clinical observations (DCO):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups. Two mid-dose male animals (No. 457 and 473) had encrusted eyes (both, reddish) during DCO days 0 - 49, respectively. One mid-dose male animal (No. 472) had an semiclosed eyelid (right) on DCO day 0. One high-dose male animal (No. 498) had a protruding eyeball (right) on DCO day 0, a protruding eyeball (right, dark red) during DCO days 7 - 14 and an injury (right, cornea bleb) during DCO days 21 - 49 (for details see 4.2.4.2.). None of these findings was considered to be treatment-related.

F1 rearing animals, Cohort 3:
All high- and most of the mid-dose males and females showed salivation during the entire study. In all these cases salivation appeared in a short time period immediately after administration and disappeared within minutes or a maximum of two hours after administration. This temporary salivation was considered to be test substance-induced. No salivation was noted in the low-dose group.
One control male animal (No. 1010) had a protruding eyeball (left) during study days 27 - 38. One high-dose female animal (No. 1133) ploughed nose first into bedding on study day 3. These observations were considered not to be associated with the test compound.
Detailed clinical observations:
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A:
There were no test substance-related mortalities in any of the groups. One female animal (No. 374) of test group 13 was sacrificed moribund because of apathy, respiration sounds, piloerection and abdominal position on study day 0. Histopathology revealed a gavage error as the most likely reason for its death.

F1 generation parental animals, Cohort 1B:
There were no test substance-related mortalities in any of the groups. One male animal (No. 475) of test group 12 and one male animal (No. 487) of test group 13 were found dead on study day 9 and study day 51, respectively. The cause of death for both decedents were most likely gavage errors.

F1 rearing animals, Cohort 3:
There were no test substance-related or spontaneous mortalities in any of the groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A:
Mean body weights and body weight change of all male and all female animals in all test substance-treated groups were comparable to the concurrent control values during the entire study. The statistically significantly increased body weight change in the mid-dose females during study days 0 - 7 was considered to be spontaneous in nature and not treatment-related since there was no relation to dose.

F1 generation parental animals, Cohort 1B:
Mean body weights and body weight change of male and female animals in all test substancetreated groups were generally comparable to the concurrent control values during the entire study.
There were statistically significant decreases in body weight/body weight change in F1B animals of various dose groups, which were small, temporary and rather inconsistent and thus considered to be of no toxicological relevance:
• decreased body weight change in high-dose males on study days 21 - 28, 35 - 42 and 42 - 49
• decreased body weight change in mid-dose males on study days 21 – 28 and 35 - 42

F1 rearing animals, Cohort 3:
The mean body weights and body weight change of all test substance-treated male and female animals were comparable to the concurrent control values throughout the entire study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

F1 rearing animals, Cohort 1A:
Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

F1 generation parental animals, Cohort 1B:
Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

F1 rearing animals, Cohort 3:
Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A:
Water consumption of all male and female animals of all test substance-treated groups was generally comparable to the concurrent control values throughout the entire study. The statistically significantly increased water consumption of the mid- and high-dose females during study days 7 - 17 and 14 - 17 was most likely spontaneous in nature and not treatmentrelated.

F1 generation parental animals, Cohort 1B:
Water consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study. The statistically significantly decreased water consumption in the high-dose males during study days 49 - 52 as well as the statistically significantly increased water consumption of the highdose females during study days 35 - 38 were considered to be spontaneous in nature and not treatment-related.

F1 rearing animals, Cohort 3:
Water consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study. The statistically significantly decreased water consumption in the mid-dose females during study days 21 - 24 was considered to be spontaneous in nature and not treatment-related since there was no relation to dose.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 rearing animals:
Cohort F1A:
No treatment-related, adverse change among hematology parameters was observed. At the end of the administration period, in males and females of test group 13 (150 mg/kg bw/d) absolute reticulocyte counts were significantly increased. Both values were above the historical control range (absolute reticulocytes, males 138.2-186.4 Giga/L; females 138.4-188.7 Giga/L). Neither any histologic finding in the spleen nor in the bone marrow were observed. The same was true for the isolated, but significant decrease of relative eosinophil cell counts in males of test group 13 with values below the historical control range (males, relative eosinophils 1.7-2.7 %). No other differential blood cell fraction was altered, and the absolute eosinophil counts were also not significantly changed and within the historical control range (males, absolute eosinophils 0.09-0.12 Giga/L). Therefore, the isolated reticulocyte count increases as well as the significant relative eosinophil decreases were regarded as maybe treatment related, but non-adverse (ECETOC Technical Report No. 85, 2002). In males of test groups 11 and 13 (15 and 150 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) were significantly shortened, but the values were within the historical control range (males, HQT 32.6-38.3 sec). Therefore, this change was regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A:
At the end of the administration period, in females of test group 13 (150 mg/kg bw/d) urea and inorganic phosphate levels were significantly increased. These changes were regarded as treatment related and adverse. In females of test group 12 (50 mg/kg bw/d) urea and inorganic phosphate values were already significantly higher compared to controls. Inorganic phosphate values were within the historical control range whereas urea values were slightly above this range (females, urea 5.63-6.34 mmol/L, inorganic phosphate 1.35-1.63 mmol/L). However, urea was the only changed parameter among these individuals and therefore, this alteration was regarded as treatment related, but non-adverse (ECETOC Technical Report No. 85, 2002), whereas the inorganic phosphate increase was regarded as incidental and not treatment related. In males of test group 13 (150 mg/kg bw/d) inorganic phosphate values were significantly increased whereas in females of this test group total bilirubin values were significantly decreased. However, both values were within historical control ranges (males, inorganic phosphate 1.60-2.04 mmol/L; females, total bilirubin 1.19-2.07 μmol/L). In males of test groups 11 and 12 (15 and 50 mg/kg bw/d) albumin values were significantly higher compared to controls, but the change was not dose dependent. Therefore, the alterations in this paragraph were regarded as incidental and not treatment related.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalyses (cohort F1A):
No treatment-related, adverse changes among urinalysis parameters were observed.

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio:
The sex distribution and sex ratios of live F1 pups on the day of birth and at weaning did not show substantial differences between the control and the test substance-treated groups; slight but statistically significant differences were regarded to be spontaneous in nature. All values were well in the PND 0 historical control range.

Vaginal opening:
Each female F1 pup, which was selected to become a rearing female (across all cohorts which have been taken beyond this age), was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 36. The mean number of days to reach the criterion in the control and 15, 50 and 150 mg/kg bw/d test groups was 30.6, 30.7, 30.9 and 31.7* (* = p≤0.05) days, respectively. The mean body weight on the day, when vaginal opening was recorded, amounted to 95.2 g, 96.4 g, 96.3 g and 97.8 g in test groups 00-03. The slightly higher high-dose time to puberty was mainly driven by one single female pup (194-04) which became sexually mature only on PND 43. As the average differed from control only by one day, the value was within the historical control range (29.5 – 38.8 days) and there were no corroborative findings in any endocrine sensitive tissues, no specific effect of the test compound on the timing of female puberty was assumed.

Preputial separation:
Each male F1 pup, which was selected to become a rearing male (across all cohorts which have been taken beyond this age), was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 39, the last was PND 47. The mean number of days to reach the criterion in the control and 15, 50 and 150 mg/kg bw/d test groups was 41.6, 41.3, 41.4 and 41.5 days, respectively. The mean body weight on the day, when preputial separation was recorded, amounted to 174.4 g, 173.7 g, 173.9 g and 172.1 g in test groups 00 - 03. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13 and during the re-examination on PND 20 in any male pups of all test groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 generation, rearing animals Cohort 1A:
Absolute organ weights:
- Liver (males): 110%* / 100% / 105% versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 109%** / 107%** / 113%** versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01
All other mean absolute weight parameters did not show significant differences when compared to the control group 10.

Relative organ weights
- Liver (males): 110%** / 103% / 108%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 108%** / 108%** / 117%** versus ctrl in low, mid and high dose groups, respectively
- Thymus (females): 98% / 106% / 109%* versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 10. As the thymus weight was not significantly changed in the immunotoxicity cohort 3 it was considered to be incidental.

F1 generation, rearing animals, cohort 1B
Absolute organ weights:
- Terminal body weithgt (males): 100% / 97% / 94%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 107%* / 109%** / 114%** versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01
All other mean absolute weight parameters did not show significant differences when compared to the control group 10.

Relative organ weights
- Liver (males): 102% / 101%* / 105%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 106%** / 109%** / 115%** versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 106% / 115%** / 105% versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01
The statistically significantly increased mean relative prostate weight in test group 12 was regarded to be incidental as there was no dose response relationship, the weight in test group 13 was not significantly changed and the weight in test group 12 (0.262%) was only very minimally (3rd decimal place) above historical controls (0.213 – 0.26%).

Comparison of liver weights in the F1 generation, rearing animals, cohort 1A and B with each other and with historical control data.The increased mean relative liver weights in males and females of test group 13 were regarded to be possibly treatment-related.

Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts):
Absolute and relative organ weights:
None of the mean absolute and relative weight parameters of test groups 01, 02, and 03 showed significant differences when compared to the control group 00.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A:
In the forestomach, an increased incidence of the following findings was noted:
Focus:
male: 6/20 (150 mg/kg bw/d)
female: 3/20 (150 mg/kg bw/d)
Margo plicatus thickened:
male: 6/20 (50 mg/kg bw/d); 20/20 (150 mg/kg bw/d)
female: 2/20 (50 mg/kg bw/d); 19/20 (150 mg/kg bw/d)

In the duodenum thickening of wall was seen in few test group 13 animals.
Thickening of wall:
male: 4/20 (150 mg/kg bw/d)
female: 5/20 (150 mg/kg bw/d)
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

F1 generation, rearing animals, cohort 1B:
In the forestomach, an increased incidence of the following findings was noted:Focus:
male: 4/25 (150 mg/kg bw/d)
female: 1/25 (50 mg/kg bw/d), 3/25 (150 mg/kg bw/d)
Margo plicatus thickened:
male: 6/25 (50 mg/kg bw/d); 20/25 (150 mg/kg bw/d)
female: 24/25 (50 mg/kg bw/d); 22/25 (150 mg/kg bw/d)
Thickening of wall:
male: 1/25 (150 mg/kg bw/d)
female: 1/25(50 mg/kg bw/d)
In a few animals, thickening of the wall of the duodenum was observed:
Thickening of wall:
male: 5/25 (150 mg/kg bw/d)
female: 3/25 (150 mg/kg bw/d)

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Decedents:
The animal No. 475 (male test group 12), died spontaneously 8 days after start of exposure and showed a red discoloration of the lungs. Male animal No. 487 (test group 13) died spontaneously 50 days after start of exposure; it showed a white deposition on the lungs on the right caudal lobe and at the transition to the diaphragm, as well as a thoracic cavity effusion with clear fluid). No histopathological evaluation was performed on these animals. The death was assumed to be likely a misgavage and not related to the test item.

Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts):
A single finding was noted in test group 02, it was considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A:
In the forestomach, erosion/ulcer were noted in many animals of both sexes of test group 13, correlating to the gross finding “focus”.
The forestomach also showed a thickening of the squamous cell layer, most frequently affecting the region of the margo plicatus, which was recorded as squamous hyperplasia margo plicatus, diffuse. This was seen in both test group 12 and with increased incidence and severity in test group 13 animals of both sexes. This finding correlated to the gross finding “margo plicatus thickened”. In a few cases the thickening of the squamous layer extended throughout the whole section of the forestomach, this was recorded as hyperplasia squamous cell diffuse.
This was only seen in few male animals of test group 13 and one male in test group 12. Incidences and gradings of these findings are shown in the table below.
Glandular stomach:
Multifocal degeneration/regeneration was seen in many animals of both sexes in test group 13. It was characterized by a loss of the normal architecture of the glandular mucosa and was replaced by more basophilic undifferentiated cells. This finding was especially prominent adjacent to the margo plicatus. Unlike in the F0 generation, no treatment-related effect could be detected regarding erosion/ulcer in the glandular stomach.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. No histopathological correlate to the gross finding “Thickening of wall” in the duodenum could be observed.

Decedents:
The female animal No. 374 (test group 13) which was sacrificed moribund showed a white deposition on the thymus and partly red discoloration macroscopically. Histopathologically there was slight multifocal inflammation on the pleura of the lungs and on the pericardium with bacterial colonies. On the thymus, correlating to the gross findings, it showed marked hemorrhage and slight multifocal inflammation admixed with bacterial colonies and foreign material. The inflammation extended to the surrounding tissue of the thyroid gland where bacterial colonies were also found. The cause of death was therefore assumed to be a gavage accident.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

F1 generation, cohort 3 animals (immunotoxicity cohort) and animals of the positive control:

T-cell dependent antibody response (cohort 3):
No treatment-related changes regarding the anti-SRBC IgM antibodies occurred in the F1 test groups at PND 63 whereas in males and females of the positive control group with administered cyclophosphamide (test group 14) a significant decrease of anti-SRBC IgM antibodies was observed.

Weight parameters
Hydroxypropyl acrylate:
Absolute and relative organ weights: None of the mean absolute and relative weight parameters of test groups 11, 12, and 13, showed significant differences when compared to the control group 10.
Cyclophosphamide monohydrate
Absolute and relative organ weights: When compared to the control group 10 (set to 100%), the mean absolute and relative weight parameters of test group 14 (positive control) were significantly decreased.
Spleen:
male: 64%** (absolute) / 67%** (relative)
female: 73%** (absolute) / 74%** (relative)
Thymus:
male: 71%** (absolute) / 74%** (relative)
female: 68%** (absolute) / 68%** (relative)
*p <= 0.05; **p <= 0.01
A significant decrease in absolute and relative weights of the spleen and thymus occurred in the positive control male and female animals. This result was expected.

Gross pathology:
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

F1 generation pups/litters:
Pup number and status at delivery:
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the rat strain used in this study.

Pup viability/mortality
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99.2% / 100% / 98.6% and 99.3% in test groups 00 - 03, showing no treatment-related effect. The lactation index indicating pup survival during PND 4 - 21 varied between 99.2% / 100% / 99.6% and100% in test groups 00 - 03, showing no treatment-related effect. Thus, the test substance did not influence pup survival in any of the treated groups (01 - 03).

Pup clinical observations:
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
For one male pup (No. 1) of test group 02 (dam No. 166) reduced nutritional condition (grade: severe) was observed on PND 7. For one female pup (No. 8) of test group 00 (dam No. 123) unsteady gait (grade: severe, abnormal position, left forelimb), semiclosed eyelid (both), reduced nutritional condition (grade: severe), piloerection and hunched posture was recorded during PND 14 - 19. These observations were considered not to be associated with the test compound.
Pup body weight data:
The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.

Pup necropsy observations in culled pups and decedents:
A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, discolored liver lobe and empty stomach. These findings occurred without any relation to dosing. Thus, all these findings were not considered to be associated to the test substance.

F1 rearing animals, Cohort 1A:
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups 10 - 13. The mean estrous cycle duration was comparable between the groups: 4.0 / 3.9 / 4.0 and 4.1 days in test groups 10 - 13, respectively.

Thyroid hormones (surplus pups PND4, PND22 and cohort F1A):
In F1 PND4 male and female pups (test groups 01, 02 and 03; 15, 50 and 150 mg/kg bw/d) no treatment related alterations of T4 and TSH levels were observed. In F1 PND22 male and female pups (test groups 01, 02 and 03; 15; 50 and 150 mg/kg bw/d) no treatment related alterations of T4 and TSH levels were observed. In cohort F1A males and females of test groups 11, 12 and 13 (15, 50 and 150 mg/kg bw/d) no treatment related alterations of TSH values as well as in males of the mentioned test groups no change of the T4 values were observed. In F1A females of test group 13 (150 mg/kg bw/d) T4 values were significantly increased, but the values were within the historical control range (F1A females, T4 26.66-60.54 nmol/L). Therefore this alteration was regarded as incidental and not treatment related.

Lymphocyte subpopulations in spleen (cohort F1A):
No alterations in the absolute and relative lymphocyte subpopulation cell counts in the spleen tissue (B-, T-lymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells) were observed in the F1 generation at PND90 in both sexes.

Spermanalysis (cohort F1A):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epidi-
dymidis as well as sperm head counts in the testis and in the cauda epididiymidis, no treatmentrelated effects were observed.

Differential ovarian follicle count (cohort F1A):
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and test group 13.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Dose descriptor:

NOAEL

Remarks:

developmental immunotoxicity

Generation:

F1 (cohort 3)

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Reproductive effects observed:

no

Conclusions:

Under the conditions of the present extended 1-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 50 mg/kg bw/d for the F0 parental as well as F1 adolescent animals, based on changed clinical-pathological parameters, which were observed at the LOAEL (Lowest Observed Adverse Effect Level) of 150 mg/kg bw/d. The NOEL (no observed effect level) for this study is below 15 mg/kg bw/d, based on clinical and pathological evidence of distinct local toxicity in the upper digestive tract, at all tested dose levels. These local effects were definitely dose-limiting.

The NOAEL for fertility and reproductive performance for the F0 parental rats is 150 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity in the F1 progeny is 150 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental immunotoxicity for the F1 progeny is 150 mg/kg bw/d, the highest dose tested. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral) demonstrated that the test system worked properly.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Chales River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France
- Age at study initiation: 11 weeks (male animals); 10 weeks (female animlas)
- Housing: During the pretreatment period of the study, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Ho
henpeißenberg, Germany. During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
- Diet (e.g. ad libitum): ad libitum (ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance solutions in drinking water were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and intensely mixed with a magnetic stirrer.

Details on mating procedure:

In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1"

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Based on the analytical results it is concluded that the substance is stable in drinking water over a period of 7 days at ambient temperature. All determined concentrations were in the range of 90% - 110% of the nominal concentration.

Duration of treatment / exposure:

After the acclimatization period, the test substance was administered to the parental animals orally by gavage.

Frequency of treatment:

once daily at approximately the same time in the morning

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity,
pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions: Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals). Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20. Food consumption of the females which gave birth to a litter was determined for PNDs
1-4, 4-7, 7-10 and 10-13. Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

Body weight data
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume;

Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals prior to the
administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

Functional observational battery
A functional observational battery (FOB) was performed in the first five male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: posture, tremors, convulsions, abnormal movements, gait, other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/color), rearing within 2 minutes, other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test

Motor activity assessment
The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For the duration of the measurement the animals were placed in new clean polycarbonate cages with a small amount of bedding. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed into the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

Clinical Pathology
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, mean corpuscular volume, mean corpuscular Hemoglobin, mean corpuscular Hemoglobin concentration, Platelet count, Differential blood, Reticulocytes, Prothrombin time.

Clinical chemistry
Parameters: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase), γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ- glutamyl-transferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, choloesterol, bile acids.

Thyroid Hormones
Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling.
All generated serum samples were frozen at -80°C until measurement.
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4).
T4 ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Oestrous cyclicity (parental animals):

For all females in a pool of 50 animals, estrous cycle normality was evaluated before randomization (the estrous cycle data of these individuals are not reported and can be found in the raw data). For a minimum of 2 weeks prior to mating, estrous cycle length and normality was evaluated by daily analysis of vaginal smears for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, one additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Litter observations:

Litter observation:
Litter data
Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in “Pup Necropsy observations“. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying
between PNDs 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13.

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Pup body weight data
The pups were weighed on the day after birth (PND 1) as well as on PND 4 (before
standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). In the summary tables pup body weights and pup body weight change are listed for males, females and males + females. „Runts“ were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Postmortem examinations (parental animals):

Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands (fixed) ,Uterus (with cervix).

The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus.

Organ/tissue fixation
Parental animals
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde solution or in modified Davidson`s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides (modified Davidson`s solution), Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson`s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer`s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson`s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson`s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964), Vagina.

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
All gross lesion, Adrenal glands, Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Eyes with optic nerve, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (axillary and mesenteric), Ovaries, Oviducts, Prostate gland, Peyer`s patches, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid , glands, Trachea, Urinary bladder, Uterus, Vagina.

Postmortem examinations (offspring):

On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing.

Statistics:

Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Reproductive indices:

The following indeces were determined: mating and fertility indes for both males and females, gestation index, live birth index and postimplantation loss for female.

Offspring viability indices:

The following indeces were determined: viability index, survival index, sex ration, anogenital index.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Most animals of test group 3 as well as several animals of test group 2 (150 and 50 mg/kg bw/d) show ed salivation after treatment (grade: slight to severe) at some occasions during the study period. In female animals the incidence was generally higher during gestation and lactation periods, affecting most test group 3 and several test group 2 females. These observations were considered to be treatment-related.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups. Spontaneous findings were seen in one high-dose male (No. 33) which showed red discharge in the right eyeduring mating days 2 - 4, 8 - 11 and postmating days 0 – 1 as well as one sperm positive mid-dose female (No. 121) which did not deliver F1 pups.

Further details / tables see attachment

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and body weight change of all male and female F0 generation parental animals in all test substance-treated groups showed no significant difference to the concurrent control during the entire study.
The statistically significantly lower body weight change in the mid-dose females during PND 7 - 10 was considered as spontaneous in nature and not as treatment-related.

Further details / tables see attachment

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the F0 males in all dose groups (15, 50 and 150 mg/kg bw/d) as well as low and mid-dose females was not influenced by the treatment throughout the study.
Food consumption of the high-dose F0 females was statistically significantly below the concurrent control values during the entire premating period (up to 7%) while it remained unchanged during gestation and lactation periods.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males of test group 3 (150 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significant ly reduced, but the mean was within the historical control range (males, HQT 37.4 - 40.9 sec). Therefore, this alteration was regarded as incidental and not treatment-related.

Further details / tables see attachment

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males of test groups 1 and 2 (15 and 50 mg/kg bw/d) cholesterol values were significantly changed (test group 1 lower, test group 2 higher) compared to controls, but the alteration was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatmentrelated.

Further details / tables see attachment

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observational battery (FOB)
Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations: Two male animals of dose group 3 (Nos. 33 and 34 - 150 mg/kg bw/d) showed slight (area around the
mouth was moist) and severe (mouth very wet, wet paws) salivation, respectively. All other male and female animals of all dose groups (15, 50 and 150 mg/kg bw/d) did not show any abnormalities.
Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.
Quantitative Parameters: No test substance-related impaired parameters were observed in male and female animals of all test
groups. The statistically significantly higher value of the landing foot splay test in females of dose group 2 was considered as spontaneous in nature and not treatment related.
Motor activity measurement (MA)
No treatment-related changes of motor activity data was observed in all male and female animals of all dose groups (15, 50 and 150 mg/kg bw/d) in comparison to the concurrent control group. Overall activity levels and habituation to the test environment corresponded to the age of these animals, if usual biological variation inherent in the strain of rats used for this experiment was considered.
The isolated statistically significantly decreased numbers of beam interrupts in the males of dose groups 1 and 2 during interval 12 were not related to the dose and did not influence the overall activity levels and habituation. Thus, they were not considered to be related to the test substance.

Further details / tables see attachment

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in forestomach and duodenum of male and female animals of test groups 2 and 3.
Duodenum: In the duodenum, a thickening of the mucosa characterized by an increase in villus height, was observed, which correlated to the macroscopic finding “dilation”. One female animal of test group 3 showed a focal hyperplasia of the duodenal mucosa.
Forestomach: Diffuse squamous hyperplasia (correlating with the macroscopic findings “margo plicatus, thickened”) and the presence of erosion/ulcer (correlating often with the macroscopic finding “focus”) were noted in the forestomach of male and female animals.
No treatment-related findings were noted in the glandular stomach and no correlate was found for the macroscopically observed discoloration in two test group 3 male animals. All other findings occurred either individually or were biologically equally distributed over control
and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.The morphology of the testicular interstitium and the stages of spermatogenesis in the testes of males of the high dose (150 ppm) were comparable to those of the controls.

Further details / tables see attachment

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was similar: 4.0 days in test groups 0 - 3, respectively.

Further details / tables see attachment

Reproductive performance:

no effects observed

Description (incidence and severity):

Further details / tables see attachment

Thyroid hormones:
In the F0 parental males of test groups 2 and 3 (50 and 150 mg/kg bw/d) T4 values were significantly higher compared to controls. However, the means were within the historical control range (males T4 44.87-88.29 nmol/L). Additionally, neither a change of thyroid weight nor any histopathological findings in the thyroids were noted. Therefore, the higher T4 values in parental males were regarded as incidental and not treatment-related.
In male and female pups at PND13 (test groups 11, 12 and 13; 15, 50 and 150 mg/kg bw/d), no treatment-related alterations of T4 levels were observed.

Further details / tables see attachment

Male reproduction data:
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in test groups 0 - 3. Fertility was proven for most of the F0 parental males within the scheduled mating interval for
F1 litter.
One mid-dose male (50 mg/kg bw/d - No. 21) did not generate pregnancy.
Thus, the male fertility index ranged between 90% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until copulation was confirmed (GD 0) varied between 2.1 and 5.3 days without showing a statistically significant difference. The reason for the higher average in test group 3 were 3 mating pairs with copulation confirmed after 12 or 14 days of pairing, respectively. All 3 affected females had normal estrous cycles and normal pregnancies, and no findings were noted in the reproductive organs of the affected males and females. The very extensive period before copulation fits best to artificial pseudo-pregnancies provoked by the daily manipulations during vaginal lavage. None of this is considered to be treatment-related.
All female rats delivered pups or had implants in utero with the following exceptions: Mid-dose female No. 121 (mated with male No. 21) did not become pregnant.
The fertility index varied between 90% in test group 2 and 100% in the control and test groups 1 and 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The non-pregnant female had no relevant gross lesions or microscopic findings.
The mean duration of gestation values varied between 21.9 (test group 1), 22.2 (control and test group 2) and 22.4 (test group 3), not indicating any influence by the test substance.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.8 / 13.0 / 13.2 and 14.3 implants/dam in test groups 0 - 3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.1 / 12.6 / 12.6 and 13.9 pups/dam in test groups 0 - 3, respectively).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.2% / 100% / 100% and 99.3% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
Thus, the test substance Hydroxypropylacrylate did not adversely affect reproduction and delivery of the F0 generation parental animals.

Further details / tables see attachment

Key result

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Key result

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed.

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: irritation in forestomach and duodenum

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

PND 0: 1 male pup died in test group 0 and 3
PND 1-4: 1 male and 1 female pup died in test group 1; 2 male pups died in test group 2

Further details / tables see attachment

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related influence on body weights and body weight change values of F1 pups were noted in test groups 1 - 3 (15, 50 and 150 mg/kg bw/d).
One female runt was seen in the control, one male and two female runts were seen in test group 1, three male and two female runts were seen in test group 2 and one male and two female runts were seen in test group 3. These are spontaneous findings unrelated to the treatment.

Further details / tables see attachment

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, discolored testis (left, red) and dilated renal pelvis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

Further details / tables see attachment

Litter data
Pup number and status at delivery
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
Pup viability
The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 100%/ 98.6% /97.9% and 100% in test groups 0 - 3, respectively. The survival index indicating pup mortality until mid-lactation (PND 4 - 13) was 100% in all test groups.
Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Anogenital distance/anogenital index
No test substance-related effects were noted on anogenital distance or anogenital index in all treated F1 offspring (test groups 1 - 3 [15, 50 and 150 mg/kg bw/d]).
Nipple/ areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Further details / tables see attachment

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

The results in tabular form are provided in the attached background material as pdf.

Reference 4

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The available data on 2-hydroxyethyl acrylate (NOAEL 120 mg/kg bw/d) provides reliable screening information for this endpoint, with no concerns identified. In addition 2-hydroxypropyl acrylate was tested in a reliable screening test ( NOAEL 150 mg/kg bw) and an extended one generation reproduction toxicity study according OECD TG 443 (NOAEL 150 mg/kg bw/d (fert./dev); NOAEL general syst. toxicity 50 mg/kg bw/d). Furthermore, long-term reproductive toxicity test data on the respective hydrolysis products of HEA and HPA (AA, EG and PG) show no reproductive toxicity effects (NOAEL 460 [2-gen], >1000 [3-gen] and >10000 [2-gen] mg/kg bw/day, respectively).
In addition to the 2-generation studies on MA and AA that were included in the dossier, an EOGRTS on nBA (Charles River, 2017) yielding a NOAEL=150 mg/kg bw/day showing no reproduction toxicity..
Therefore, overall the acrylates show a non-reprotoxic profile as evidenced by the test results on HPA, nBA, and MA, as well as the common hydrolysis product AA. The non-common hydrolysis product (EG) specific to HEA also does not exhibit reproductive toxicity effects.Taken together, these two lines of evidences allow us to conclude that HEA does not have the intrinsic ability to produce reproductive toxicity effects
Based on these data, we propose to fill the requirement of EOGRTS for HEA by reading across to the studies on MA (2-generation) and nBA (EOGRTS) and the data on the hydrolysis products of HEA (AA and EG) as supporting information. . This approach is further documented in the attached read-across justification.

Please see for more information the read-across justification in Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOEC

Remarks:

reproductive toxicity / rat

Effect level:

ca. 0.269 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 75 ppm; the highest concentration tested.

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

rat_2009 / Correction for molecular weight not necessary.

Dose descriptor:

NOEC

Remarks:

parental local toxicity / rat

Effect level:

ca. 0.019 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 5 ppm; based on histologic changes in nasal tissues.

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

rat_2009 / Correction for molecular weight not necessary

Dose descriptor:

NOAEL

Remarks:

systemic toxicity / rat

Effect level:

240 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

Remarks on result:

other: Result read-across CAS No. 79-10-7

Remarks:

rat_1994 / Correction for molecular weigt not necessary

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity / rat

Effect level:

460 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Result read-across CAS No. 79-10-7

Remarks:

rat_1994 / Correction for molecular weight not necessary.

Key result

Critical effects observed:

no

Dose descriptor:

NOEC

Remarks:

parental local toxicity / rat

Effect level:

ca. 0.019 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: corresponding to 5 ppm; based on histologic changes in nasal tissues.

Remarks on result:

other: corresponding to 5 ppm; Result read-across CAS No. 96-33-3

Remarks:

rat_2009 / Correction for molecular weight not necessary.

Key result

Dose descriptor:

NOEC

Remarks:

reproductive toxicity / rat

Effect level:

ca. 0.269 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 75 ppm; the highest concentration tested.

Remarks on result:

other: corresponding to 5 ppm; Result read-across CAS No. 96-33-3

Remarks:

rat_2009 / Correction for molecular weight not necessary.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity / rat

Effect level:

440 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Result read-across CAS No. 79-10-7

Remarks:

rat_1994 / Correction for molecular weight not necessary.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity / rat

Effect level:

53 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

Remarks on result:

other: Result read-across CAS No. 79-10-7

Remarks:

rat_1994 / Correction for molecular weight not necessary.

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOEC

Remarks:

rat

Generation:

F1

Effect level:

ca. 0.092 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 25 ppm; based on decreases in pup body weights at 75 ppm which were secondary to parental toxicity.

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

rat_2009 / Correction for molecular weight not necessary

Key result

Dose descriptor:

NOAEL

Remarks:

rat

Generation:

F1

Effect level:

53 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Result read-across CAS No. 79-10-7

Remarks:

rat_1994 / Correction for molecular weight not necessary.

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOEC

Remarks:

rat

Generation:

F2

Effect level:

ca. 0.092 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 25 ppm; based on decreases in pup at 75 ppm which were secondary to parental toxicity.

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

rat_2009 / Correction for molecular weight not necessary.

Key result

Dose descriptor:

NOAEL

Remarks:

rat

Generation:

F2

Effect level:

53 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Result read-across CAS No. 79-10-7

Remarks:

rat_1994 / Correction for molecular weight not necessary.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

0.269 mg/L air (analytical)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

240 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Reference 5

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

28 Jul 2011

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 70 days
- Basis for dose level selection: Dosage levels were selected based on the range-finding study. In that study, dosage levels of 40, 160, and 400 mg/kg/day were tested. Animals in the high-dosage group showed clinical signs (salivation and red material around the eyes, nose, and mouth), which were still observed to a lesser extent in the mid-dosage group, and the males were more affected than the females. Macroscopic examinations showed thickened and eroded stomach in the 400 mg/kg/day group males and thickened stomach was still observed in the 160 mg/kg/day group animals (predominantly in males). In the current study, the animals were dosed for a longer time period. Therefore, based on the results of the range-finding study, dosage levels of 20, 50, and 150 mg/kg/day were selected for the current study. The high-dosage level of 150 mg/kg/day was expected to show parental toxicity, whereas the low-dosage level of 20 mg/kg/day was not expected to show toxic effects.
- Inclusion/exclusion of extension of Cohort 1B: not performed as P0 and F1 Cohort 1A did not reveal any results
- Termination time for F2: not performed as P0 and F1 Cohort 1A did not reveal any results
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: not performed as P0 and F1 Cohort 1A did not reveal any results
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: not performed as P0 and F1 Cohort 1A did not reveal any results
- Route of administration: oral

Specific details on test material used for the study:

- Lot No. F534801GB
- Exp. date: 11 Dec 2016
- Colorless, clear liquid

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 8 weeks
- Weight at study initiation: Male body weights ranged from 264 g to 378 g and female body weights ranged from 154 g to 228 g on the initial day of test substance administration
- Housing: F0 parental animals were housed 2–3 per cage by sex in clean, solid-bottom cages with heat-treated aspen bedding material (Aspen Bed™ 1, Northeastern Products Corporation, Warrensburg, NY). Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly
- Diet: the basal diet was provided ad libitum throughout the acclimation period and during the study. The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5K96 Advanced Protocol® Verified Casein Diet 10 IF, was a certified feed with appropriate analyses performed by the manufacturer.
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system was provided ad libitum throughout the acclimation period and during the study.
- Acclimation period: 24 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: 1% carboxymethylcellulose (medium viscosity), 0.014% Kolliphor® EL, and 0.0035% hydrochloric acid in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared daily as single formulations for each dosage level and maintained on wet ice, protected from light. The test substance formulations were stirred continuously on wet ice throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 4, 10 and 30 mg/mL (corresponding to dosage levels of 0, 20 50 and 150 mg/kg/day)
- Amount of vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: The animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of treatment.
- Length of cohabitation: 14 day mating period
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Following positive evidence of mating, the F0 males and females were individually housed in solid-bottom cages with bedding material until the scheduled necropsy. The dams were housed in these cages with their litters until weaning on Lactation Day 21. Following weaning of the F1 litters, the weaned F1 pups were housed together by litter for 1 week. Beginning on PND 28, the F1 offspring were housed 2–3 per cage by sex in clean, solid-bottom cages with bedding material until necropsy. Females for which there was no evidence of mating were placed in clean, solid-bottom cages with bedding material upon completion of a 14-day mating period with no further opportunity for mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the 4, 10, and 30 mg/mL dosing formulations and from the middle stratum of the control group dosing formulations prepared on Study Day 0, 7, 15, and 30 for the first month and once a month thereafter (total of 9 sets). Analysis was performed using a validated gas chromatography method using flame ionization detection. The analysed dosing formulations were, with minor exceptions, within the range for suspensions (85% to 115%) and were homogeneous. Analysed concentrations above the acceptance criteria had no impact on the study as the no-observed-adverse-effect level (NOAEL) was determined to be 150 mg/kg/day, the highest dose evaluated on this study.

Duration of treatment / exposure:

The vehicle and test substance formulations were administered to the F0 males and females for a minimum of 70 consecutive days prior to mating. Dose administration for the F0 males continued throughout mating and through the day prior to euthanasia, for a total of 129-131 doses. The F0 females continued to be dosed throughout mating, gestation, and lactation, through the day prior to euthanasia, for a total of 132-136 doses. Animals selected for the F1 generation were directly administered the vehicle or test substance following weaning (beginning on PND 21) and continuing through the day prior to euthanasia (PND 91 [Cohort 1A] and between PND 103-110 [Cohort 1B]).

Frequency of treatment:

daily

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Details on study design:

Dosage levels were selected based on the range-finding study (see any other information on materials and methods)

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS
Detailed physical examinations were recorded weekly, beginning 1 week prior to the initiation of test substance administration, for all parental animals throughout the study period. In addition, detailed physical examinations were conducted on Gestation Days 0, 7, 14, and 20 for all females with evidence of mating and on Lactation Days 1, 7, 14, and 21. In addition, animals were observed for signs of toxicity approximately 1 hour following dose administration. Special attention was paid to the degree of salivation and lacrimation, presence or absence of urination and defecation (including polyuria and diarrhoea), pupil size, degree of palpebral closure, presence of convulsions, tremors, or abnormal movements, presence of posture and gait abnormalities, the presence of any unusual or abnormal behaviours, and any repetitive actions (stereotypies). Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labour, delayed labour) or other difficulties. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal.

BODY WEIGHT
Individual F0 male body weights were recorded weekly, beginning 1 week prior to the initiation of test substance administration, throughout the study and prior to the scheduled necropsy. Individual F0 female body weights were recorded weekly, beginning 1 week prior to the initiation of test substance administration, until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating treatment period (males and females) and for the entire F0 treatment period (males only). Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, 14, 17, and 21. After weaning (Lactation Day 21), weekly body weights were recorded for these females until the scheduled necropsy. For F0 females with no evidence of mating, weekly body weights were recorded until necropsy.

FOOD CONSUMPTION
F0 male and female food consumption was measured weekly, beginning 1 week prior to the initiation of test substance administration and continuing until cohabitation. Food consumption was measured on a per cage basis for the corresponding body weight intervals. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food intake was not recorded during the breeding period. Following the breeding period, individual food consumption for males and for females with no evidence of mating was measured on a weekly basis until the scheduled necropsy. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 14, 17, and 21. Food intake was reported for the corresponding intervals of gestation and lactation, and also for Gestation Days 0–20 and Lactation Days 1–21. Food efficiency (body weight gained as a percentage of food consumed) was also calculated

CLINICAL PATHOLOGY
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected prior to the scheduled necropsy (Study Days 129-136 for the F0 males and females). All animals were fasted overnight prior to blood collection while in metabolism cages for urine collection blood and urine samples were collected from 10 randomly selected F0 animals/sex/group sent to necropsy. Blood for serum chemistry and haematology was collected from the jugular vein using the hand-held technique following anaesthesia with isoflurane inhalation. Blood for coagulation parameters was collected from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.

- Haematology and coagulation parameters examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (Platelet), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC Absolute), Mean platelet volume (MPV), Differential leukocyte count Percent and absolute, Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Red cell distribution width (RDW), Haemoglobin distribution width (HDW), Platelet estimate, Red cell morphology (RBC Morphology).
- Serum chemistry parameters examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total BILI), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyl transferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Appearance, Bile Acids
- Urinalysis parameters examined: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Colour (COL), Clarity (CLA), Protein (PRO), Glucose (GLU)

THYROID HORMONE ANALYSIS
Blood (approximately 1.5 mL) for thyroid hormone levels was collected via the retro-orbital sinus following isoflurane anaesthesia from F0 males and females (10/sex/group) following the completion of weaning of all F0 females
- Parameters evaluated: Thyroxine (T4) and Thyroid Stimulating Hormone (TSH)

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each F0 female for 14 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete oestrous cycles (i.e., the total number of returns to metoestrous [M] or dioestrous [D] from oestrus [E] or prooestrous [P], beginning 14 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual oestrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the oestrous cycle. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.

Sperm parameters (parental animals):

Immediately upon euthanasia, the reproductive tract of each F0 male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed separately. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a 10 to 20-minute incubation period, a sample of sperm was loaded onto a slide with a 100-μm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all slides and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyser. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:
Percent Motile (or Progrressively Molite) Sperm = Number of Motile (or Progressively Motile) Sperm / Total Number of Sperm Counted x 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all F0 males from the control and high-dose groups were weighed, stored frozen, homogenized, and analysed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the analyser allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = No. of Sperm Per Gram of Tissue / 6.1 days
6.1 days = The rate of turnover of the germinal epithelium

Litter observations:

STANDARDISATION OF LITTERS
To reduce variability among the litters, 10 pups/litter, 5 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 10 pups. All selections were performed by computerized randomization. Following selection, culled pups were euthanized by decapitation (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital, and examined macroscopically; tissues with gross lesions were preserved in 10% neutral-buffered formalin.

PARAMETERS EXAMINED PRE-WEANING
- A daily record of litter size was maintained. Intact offspring that were found dead during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels.7 Pups found dead on PND 0 or 1 had the lungs removed and placed in a saline-filled jar. If the lungs sank to the bottom of the jar, the pup had no prior documentation of being viable, and there was no evidence of milk in the stomach, the pup was considered to be stillborn. If the lungs floated, the pup was considered to be found dead on the originally documented day (PND 0 or 1).
- Litters were examined daily for survival and any adverse changes in appearance or behaviour. Each pup received a detailed physical examination on PND 1, 4, 7, 14, and 21.
- Pups were individually weighed on PND 1, 4 (before culling), 7, 14, and 21.
- Pups were individually sexed on PND 0, 4 (before culling), 7, 14, and 21.
- The anogenital distance of all F1 pups was measured on PND 1
- On PND 12, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae.

GROSS EXAMINATION OF DEAD PUPS
A detailed gross necropsy was performed on any pup found dead after PND 4 and prior to weaning. Tissues were preserved for possible future histopathological examination only as deemed necessary by the gross findings.

POST-WEANING DEVELOPMENTAL LANDMARKS
- Balanopreputial Separation: Each male was observed for balanopreputial separation beginning on PND 35. The age at which balanopreputial separation was first observed was recorded for each pup. Examination of the pups continued daily until balanopreputial separation was present. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a partial and complete balanopreputial separation or a persistent thread of tissue between the glans and prepuce was recorded.
- Vaginal Patency: Each female was observed for vaginal perforation beginning on PND 25. The age at which the vaginal lumen was first observed to open was recorded for each pup. Examination of the females was continued daily until vaginal patency was present. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a small “pin hole”, a vaginal thread, and complete vaginal opening were recorded.
- Oestrous Cyclicity (Cohort 1A): Beginning on the day vaginal opening was observed, vaginal lavages were performed daily for all F1 females assigned to Cohort 1A and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female until the first sign of oestrus (cornified cells) was observed. The age of first vaginal oestrus after vaginal opening was recorded. Vaginal lavages were also performed daily for all F1 females assigned to Cohort 1A and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each female for 2 weeks during PND 75–91 (the day of necropsy). The average cycle length was calculated and reported for complete oestrous cycles (i.e., the total number of returns to metaoestrus [M] or dioestrus [D] from oestrus [E] or prooestrus [P]). Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.

POST WEANING OBSERVATIONS
- Clinical observations: Following weaning, all animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Detailed physical examinations were performed weekly until necropsy. In addition, animals were observed for signs of toxicity approximately 1 hour following dose administration.
- Body weights: F1 males and females were weighed weekly following weaning, and on the day of euthanasia.
- Food Consumption: F1 male and female food consumption was measured weekly, beginning on PND 28, until the day prior to euthanasia. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for these intervals.

CLINICAL PATHOLOGY (Cohort 1A)
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected prior to the scheduled necropsy (PND 91 for the Cohort 1A F1 males and females). All animals were fasted overnight prior to blood collection while in metabolism cages for urine collection blood and urine samples were collected from10 randomly selected F0 and F1 Cohort 1A animals/sex/group sent to necropsy. Blood for serum chemistry and haematology was collected from the jugular vein using the hand-held technique following anaesthesia with isoflurane inhalation. Blood for coagulation parameters was collected from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.

- Haematology and coagulation parameters examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (Platelet), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC Absolute), Mean platelet volume (MPV), Differential leukocyte count Percent and absolute, Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Red cell distribution width (RDW), Haemoglobin distribution width (HDW), Platelet estimate, Red cell morphology (RBC Morphology).
- Serum chemistry parameters examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total BILI), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyl transferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Appearance, Bile Acids
- Urinalysis: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Colour (COL), Clarity (CLA), Protein (PRO), Glucose (GLU)

Postmortem examinations (parental animals):

SACRIFICE
The surviving F0 males and females were euthanized on Study Days 129-136

GROSS NECROPSY
- A complete necropsy was conducted on all F0 animals that were found dead, euthanized in extremis, or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. Clinical findings that were verified at necropsy were designated CEO. For F0 females that delivered, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Numbers of corpora lutea were also recorded for females necropsied during gestation through Lactation Day 4. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
- The following F0 parental tissues and organs were collected and were placed in 10% neutral-buffered formalin: Adrenal glands (2), Aorta (1), Bone with marrow (sternebrae), Brain (7 levels), Coagulating glands (2), Eyes with optic nerve (2) [a], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Lacrimal/Harderian glands (2), Liver (sections of 2 lobes fixed and also 2 sections frozen) [b], Lungs (including bronchi, fixed by inflation with fixative) (5), Lymph node (mandibular and mesenteric) (1), Ovaries [c] and oviducts (2), Pancreas (1), Peripheral nerve (tibial), Pituitary (1), Prostate (1), Mandibular salivary gland (1), Seminal vesicles (2), Skeletal muscle (rectus femoris) (1), Skin with mammary gland and subcutis (1)[d], Spinal cord (cervical, thoracic, lumbar) (3), Spleen (1), Testes with epididymides (1) and, vas deferens (1) [e,f],Thymus, Thyroids with trachea [g] [with parathyroids if present (1)], Urinary bladder, uterus with cervix and vagina (4), All gross lesions (all groups).
The number in parentheses is the minimum number of sections to be examined if the tissue was chosen for histopathologic examination.
[a] Eyes with optic nerves were fixed in Davidson’s solution.
[b] Sections of 2 liver lobes were placed in 10% neutral-buffered formalin and 2 sections of liver lobes were flash frozen in liquid nitrogen and stored frozen at -70°C for possible future analysis.
[c] Ovaries were fixed in 10% neutral-buffered formalin for approximately 48 hours following which all ovaries were transferred to 70% ethanol. The ovaries were processed to blocks.
[d] For females, a corresponding section of skin was taken from the same anatomic area for males.
[e] Testis (right only) and epididymides (right and left caput and body) were fixed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
[f] If the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson’s solution and the right testis and epididymis were homogenized
[g] Thyroids and parathyroids (if present) were removed from the trachea following fixation to ensure tissue structure was maintained during prosection.

ORGAN WEIGHTS
Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported. The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides (total and cauda) [a], Heart, Kidneys, Levator ani and bulbocavernosus (LABC) muscle group, Liver, Ovaries, Pituitary gland, Prostate gland, Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes [a], Thyroids with parathyroids [b], Thymus gland, Uterus with oviducts and cervix.
[a] These paired organs were weighed separately.
[b] Tissues were weighed after fixation in 10% neutral-buffered formalin.

HISTOPATHOLOGY
Microscopic examination was performed on all tissues from all F0 parental from the control and high-dose groups and for all adult animals found dead and euthanized in extremis. In addition, reproductive organs of all F0 animals suspected of reduced fertility (e.g., those that failed to mate, conceive, sire, or deliver healthy offspring, or for which oestrous cyclicity or sperm number, motility, or morphology were affected) were subjected to histopathological examination. Additionally, all gross lesions were examined microscopically, irrespective of group.

Postmortem examinations (offspring):

SACRIFICE
- Euthanasia was scheduled PND 91 [Cohort 1A] and between PND 103-110 [Cohort 1B]
- All nonselected F1 weanlings were euthanized on PND 21.
- These animals were subjected to post-mortem examinations (macroscopic and microscopic examination) as follows:

GROSS NECROPSY
- A complete necropsy was conducted on all F1 animals in Cohorts 1A and 1B that were found dead, euthanized in extremis, or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. Clinical findings that were verified at necropsy were designated CEO.
- The following F1 Cohorts 1A and 1B tissues and organs were collected and were placed in 10% neutral-buffered formalin: Adrenal glands (2), Aorta (1), Bone with marrow (sternebrae), Brain (7 levels), Coagulating glands (2), Eyes with optic nerve (2) [a], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Lacrimal/Harderian glands (2), Liver (sections of 2 lobes fixed and also 2 sections frozen) [b], Lungs (including bronchi, fixed by inflation with fixative) (5), Lymph node (mandibular and mesenteric) (1), Ovaries [c] and oviducts (2), Pancreas (1), Peripheral nerve (tibial), Pituitary (1), Prostate (1), Mandibular salivary gland (1), Seminal vesicles (2), Skeletal muscle (rectus femoris) (1), Skin with mammary gland and subcutis (1)[d], Spinal cord (cervical, thoracic, lumbar) (3), Spleen (1), Testes with epididymides (1) and, vas deferens (1) [e,f],Thymus, Thyroids with trachea [g] [with parathyroids if present (1)], Urinary bladder, uterus with cervix and vagina (4), All gross lesions (all groups).
The number in parentheses is the minimum number of sections to be examined if the tissue was chosen for histopathologic examination.
[a] Eyes with optic nerves were fixed in Davidson’s solution.
[b] Sections of 2 liver lobes were placed in 10% neutral-buffered formalin and 2 sections of liver lobes were flash frozen in liquid nitrogen and stored frozen at -70°C for possible future analysis.
[c] Ovaries were fixed in 10% neutral-buffered formalin for approximately 48 hours following which all ovaries were transferred to 70% ethanol. The ovaries were processed to blocks.
[d] For females, a corresponding section of skin was taken from the same anatomic area for males.
[e] Testis (right only) and epididymides (right and left caput and body) were fixed in modified Davidson’s solution. Both testes and epididymides from animals euthanized in extremis and from all males in Cohort 1B were fixed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
[f] If the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson’s solution and the right testis and epididymis were homogenized
[g] Thyroids and parathyroids (if present) were removed from the trachea following fixation to ensure tissue structure was maintained during prosection.
- Gross necropsies with emphasis on developmental morphology and organs of the reproductive system were performed on all nonselected F1 pups euthanized on PND 21.
-The following tissues/organs from 10 F1 nonselected pups/sex/group were retained in 10% neutral-buffered formalin for possible histopathologic examination: Brain, Mammary gland, Spleen, Thymus, Thyroids, All gross lesions

ORGAN WEIGHTS
- Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported. The following organs were weighed from all F1 animals in Cohort 1A at the scheduled necropsies: Adrenal glands, Brain, Epididymides (total and cauda) [a,c], Heart, Kidneys, Levator ani and bulbocavernosus (LABC) muscle group[c], Liver, Ovaries [c], Pituitary gland [c], Prostate gland [c], Seminal vesicles with coagulating glands (with accessory fluids) [c], Spleen, Testes [a,c], Thyroids with parathyroids [b], Thymus gland, Uterus with oviducts and cervix [c]
[a] These paired organs were weighed separately.
[b] Tissues were weighed after fixation in 10% neutral-buffered formalin.
[c] Also weighed for F1 Cohort 1B animals.
- The brain, spleen and thymus were weighed from 10 nonselected F1 pups/sex/group on PND 21. In addition, the thyroids were weighed (after fixation) from the same 10 pups/sex/group that were used for blood collection

HISTOPATHOLOGY
- Microscopic examination was performed on all tissues from all F1 Cohort 1A animals from the control and high-dose groups and for all adult animals found dead and euthanized in extremis. Additionally, all gross lesions were examined microscopically, irrespective of group.

SPERM PARAMETERS (Cohort 1A)
Immediately upon euthanasia, the reproductive tract of each F1 Cohort 1A male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed separately. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a 10 to 20-minute incubation period, a sample of sperm was loaded onto a slide with a 100-μm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all slides and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyser. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:
Percent Motile (or Progrressively Molite) Sperm = Number of Motile (or Progressively Motile) Sperm / Total Number of Sperm Counted x 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all F1 Cohort 1A males from the control and high-dose groups were weighed, stored frozen, homogenized, and analysed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the analyser allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = No. of Sperm Per Gram of Tissue / 6.1 days
6.1 days = The rate of turnover of the germinal epithelium

Statistics:

see any other information on materials and methods

Reproductive indices:

- Male Mating Index (%) = No. of Males with Evidence of Mating / Total No. of Males Used for Mating x 100
- Female Mating Index (%) = No. of Females with Evidence of Mating or Females Confirmed Pregnant / Total No. of Females Used for Mating x 100
- Male Fertility Index (%) = No. of Males Siring a Litter/ Total No. of Males Used for Mating x 100
- Male Copulation Index (%) = No. of Males Siring a Litter/ No. of Males with Evidence of Mating (or Females Confirmed Pregnant) x 100
- Female Fertility Index (%) = No. of Females with Confirmed Pregnancy/ Total No. of Females used for Mating x 100
- Female Conception Index (%) = No. of Females with Confirmed Pregnancy/ No. of Females with Evidence of Mating (or Females Confirmed Pregnant) x 100

Offspring viability indices:

- Mean Live Litter Size = Total Viable Pups on PND 0/ No. Litters with Viable Pups on PND 0
- Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection)(% Per Litter) = Sum of (Viable Pups/Litter on PND 0 or PND 4 [Pre-Selection]/No. of Pups Born/Litter)/ No. of Litters/Group x 100
- Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups/Litter at End of Interval N/Viable Pups/Litter at Start of Interval N)/ No. of Litters/Group x 100

Where N = PND 0–1, 1–4 (Pre-Selection), 4 (Post-Selection)–7, 7–14, 14–21, Birth to PND 4 (Pre-Selection), or 4 (Post-Selection)–21

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related clinical observations were noted during the F0 generation at the daily examinations, weekly detailed physical examinations, and/or approximately 1 hour following dose administration. Clonic convulsions were noted for 1 male in the 20 mg/kg/day group, 1 female in the 50 mg/kg/day group, and 1 male and 1 female in the 150 mg/kg/day group sporadically throughout the treatment period; this finding was noted at the daily examinations, weekly detailed physical examinations, at the time of dose administration, and/or approximately 1 hour following dose administration. This finding was transient and was not observed in the F1 generation, and therefore was not considered test substance-related. Other observations noted in the test substance-treated groups, including red and/or yellow material, scabbing, and/or hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test substance-related effects on survival were noted for F0 males and females at any dosage level. One female (in the 50 mg/kg/day group) was euthanized in extremis on Gestation Day 26 due to poor general health. The cause of morbidity was the acute inflammation of the uterus observed microscopically and the partially decomposed fetuses observed grossly. Because all animals in the high-dose group (150 mg/kg/day) survived to the scheduled necropsy, the moribundity in the 50 mg/kg/day group was not considered test substance-related. All other F0 parental animals in the control, 20, 50, and 150 mg/kg/day groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related effects on F0 mean body weights, body weight gains, and cumulative body weight gains were noted in the 20, 50, and 150 mg/kg/day groups. Differences from the control group were not statistically significant, with the following exceptions. For F0 males, significantly (p < 0.01) lower mean body weight gains were noted in the 20 mg/kg/day group during Study Days 112–119 and in the 150 mg/kg/day group during Study Days 91–98 compared to the control group; these differences were transient, did not occur in a dose-related manner, and/or were not of sufficient magnitude to affect the entire generation interval (Study Days 0–126) or mean body weights in these groups, and therefore were not considered test substance-related. A significantly (p < 0.05) higher mean body weight was noted for the 150 mg/kg/day group F0 females compared to the control group on Study Day 14; this difference was transient and no corresponding effect on mean body weight gain was noted in this group, and therefore was not considered test substance-related.
- Mean F0 maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during gestation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant, with the following exception. A significantly (p < 0.05) lower mean body weight gain was noted in the 50 mg/kg/day group F0 females compared to the control group during Gestation Days 17–20; this difference was transient, did not occur in a dose-related manner, was not of sufficient magnitude to affect the overall gestation treatment interval (Gestation Days 0–20) or mean body weights in this group, and therefore was not considered test substance-related.
- Mean F0 maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during lactation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- Mean F0 food consumption in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. The values in the test substance-treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Differences from the control group were not statistically significant, with the following exceptions. In F0 females, significantly (p < 0.05 or p < 0.01) higher mean food consumption was noted in the 20 mg/kg/day group during Study Days 7–14 and in the 150 mg/kg/day group during Study Days 0–7 and 14–21; these differences were transient, did not occur in a dose-related manner, and/or were not of sufficient magnitude to affect mean body weights at these dosage levels, and therefore were not considered test substance-related.
- Mean F0 maternal food consumption was unaffected by test substance administration during gestation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.
- Mean F0 maternal food consumption was unaffected by test substance administration during lactation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

- Mean F0 food efficiency in the 20, 50, and 150 mg/kg/day groups were unaffected by test substance administration.
- Differences from the control group were not statistically significant, with the following exceptions. Significantly (p < 0.01) lower mean food efficiency was noted for F0 males in the 20 mg/kg/day group during Study Days 112–119 and in the 150 mg/kg/day group during Study Days 91-98 compared to the control group; these differences were transient, did not occur in a dose-related manner, and/or a corresponding effect on mean food consumption was not noted at these dosage levels, and therefore they were not considered test substance-related.
- Mean F0 maternal food efficiency was unaffected by test substance administration during gestation.
- Mean F0 maternal food efficiency was unaffected by test substanceadministration during lactation.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related alterations in serum chemistry parameters. Significantly (p < 0.05) higher mean serum albumin was observed in the 150 mg/kg/day group F0 females when compared to the concurrent control group. The mean and individual animal values were within the Charles River Ashland historical control ranges for female rats. The finding was considered a result of individual animal variation rather than a true test substance-related change.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Test substance-related microscopic findings were noted in the nonglandular stomach and liver of the 150 mg/kg/day group F0 males and females and the kidneys in the 150 mg/kg/day group F0 females.
- Nonglandular stomach findings were limited to minimal to moderate epithelial hyperplasia and hyperkeratosis. The hyperplasia was characterized by increased thickness (increased number of cell layers) of the squamous epithelium with an increased thickness of the keratinized oute layers of the epithelium (hyperkeratosis). In one 150 mg/kg/day group F0 female, minimal edema and congestion were observed in the submucosa adjacent to the hyperplasia and hyperkeratosis. These changes were not associated with any clinical pathology changes. Although considered an adaptive change, the mild to moderate severity of the hyperplasia and/or hyperkeratosis in the F0 generation were considered adverse in the 150 mg/kg/day group males and females.
- Liver changes observed microscopically included increased incidence of minimal to mild biliary hyperplasia in the 150 mg/kg/day group F0 males and females and minimal to mild randomly scattered hepatocellular necrosis in the 150 mg/kg/day group F0 males. Biliary hyperplasia was characterized by increased numbers of small bile duct profiles in the portal triads. Biliary hyperplasia can occur as a normal age related finding in rats and was observed in the concurrent control group in this study. The severity was minimal to mild and a clinical pathology correlate for the finding was not observed. Similarly, randomly distributed hepatocellular necrosis can be observed spontaneously in rats and was observed in the control group in this study. The severity of the necrosis was minimal to mild and the change lacked clinical pathology correlates. There was no association of the biliary hyperplasia to the necrosis. Changes observed in the liver were considered nonadverse.
- Increased severity of mineralization at the corticomedullary junction of the kidneys was observed in the 150 mg/kg/day group F0 females. This increase in severity (minimal to mild) did not result in alterations to the clinical pathology parameters related to renal function and were considered nonadverse.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) in F0 males or females. Differences from the control group were slight, did not occur in a dose-related manner, and/or were not statistically significant.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean lengths of oestrous cycles in the test groups were also similar to the control group value. None of these differences were statistically significant. All females were noted to be cycling, and the percentage of females cycling regularly was similar across all groups.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level. Differences from the control group were slight and were not statistically significant, with the following exceptions. Left cauda epididymal sperm concentration was significantly (p < 0.05) lower in the 150 mg/kg/day group compared to the control group. However, there were no effects on reproductive performance (mating and fertility) and no other effects on spermatogenic endpoints at this dosage level; therefore, the lower epididymal sperm concentration noted at 150 mg/kg/day was not considered test substance-related. Significantly (p < 0.05) lower mean motility and progressive motility were noted in the 20 mg/kg/day group compared to the control group; these differences did not occur in a dose-related manner, and therefore were not considered test substance-related.

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on F0 reproductive performance were observed at any dosage level. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value.

No test substance-related effects were noted on mean gestation lengths or the process of parturition at any dosage level.

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no reproductive effects observed

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Dose descriptor:

LOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effects (nonglandular stomach)

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance administration.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects on survival were noted for F1 animals at any dosage level. In the 50 mg/kg/day group, 1 female was found dead on PND 82 following clinical observations of gasping prior to and following dose administration on the day of death. Based on the findings noted for this female at necropsy (esophageal perforation and clear fluid contents in the thoracic cavity [approximately 8.0 mL]), the cause of death was determined to be intubation error, and therefore was not considered test substance-related. In the control group, 1 male was euthanized in extremis on PND 28 due to clinical observations of head tilt and circling on the day of euthanasia; at necropsy, this male was noted with dilated lateral ventricles in the brain. This correlated to the marked chronic active inflammation in the brain. All other F1 animals in the control, 20, 50, and 150 mg/kg/day groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Mean F1 male and female pup body weights and body weight changes in the 20, 50, and 150 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period.
- No test substance-related effects on mean body weights, body weight gains, and cumulative body weight gains were noted in the 20, 50, and 150 mg/kg/day group F1 males and females. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p < 0.01) lower mean body weight gain was noted in the 20 mg/kg/day group F1 males compared to the control group during PND 42-49; this difference was transient, did not occur in a dose-related manner, and was not of sufficient magnitude to affect mean body weights at this dosage level, and therefore was not considered test substance-related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean F1 food consumption in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p < 0.05 or p < 0.01) lower mean food consumption was noted in the 20 mg/kg/day group F1 males during PND 42–49 and 63–70, in the 50 mg/kg/day group F1 males during PND 70–77, and in the 50 mg/kg/day group F1 females during PND 49–63 and 70–77 compared to the control group; these differences were transient, did not occur in a dose-related manner, and were not of sufficient magnitude to affect mean body weights at these dosage levels, and therefore were not considered test substance-related.

Food efficiency:

no effects observed

Description (incidence and severity):

Mean F1 food efficiency in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- There were no test substance-related effects on serum chemistry parameters.

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Description (incidence and severity):

- Mean estrous cycle lengths in the test substance-treated groups were similar to the control group.
- No test substance-related effects were observed on F1 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level.
- There were no test substance-related effects on ovarian primordial follicle counts in the 150 mg/kg/day group F1 females in Cohort 1A compared to the control group.
- Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance administration.
- Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance administration.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed for PND 21 pups at any dosage level.
-There were no test substance-related changes in organ weights for F1 males and females in Cohort 1A. The absolute and relative pituitary weights were significantly (p < 0.05 or p < 0.01) higher in the 50 and 150 mg/kg/day group F1 females when compared to the concurrent control. These values fell within the historical control reference ranges, were not associated with any microscopic findings, and/or were considered a result of individual animal variation rather than a true test substance-related effect.

-No test substance-related effects on organ weights (absolute and relative to final body weight) were observed at any dosage level for F1 animals in Cohort 1B (necropsied on PND 103–110). Differences from the control group were slight and not statistically significant, with the following exceptions. A higher mean absolute left testis weight was noted in 50 mg/kg/day group F1 males and higher mean relative (to final body weight) left and right testis weights were noted in the 20 and 50 mg/kg/day group F1 males compared to the control group; these differences did not occur in a dose-related manner, and therefore were not considered test substance-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- The numbers of pups (litters) found dead from PND 0 through the selection of the F1 generation were 50(13), 46(14), 24(5), and 31(11) in the control, 20, 50, and 150 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration of the test substance were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach and lungs that did or did not float, internal findings included renal papilla(e) not fully developed (Woo and Hoar Grade 1) for 2 pups in the 50 mg/kg/day group and red fluid contents in the bladder for 1 pup in the 20 mg/kg/day group. Because the findings in the 20 and 50 mg/kg/day groups were noted infrequently and in a non-dose-related manner, they were not considered test substance-related.
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsy of culled pups euthanized on PND 4. Internal findings included the developmental variations of distended ureters noted for one pup in the 50 mg/kg/day group and hemorrhagic ring around the iris noted for 1 pup each in the control and 20 mg/kg/day groups. These findings occurred in single pups and in a non-dose-related manner, and therefore were not considered test substance-related.
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsy of pups euthanized on PND 21. Dilated renal pelvis was noted for 1, 2 , and 1 pups in the 20, 50, and 150 mg/kg/day groups, respectively; 1 of these same pups in the 50 mg/kg/day group was also noted with distended ureters. In the 150 mg/kg/day group, 1 pup was noted with opacity of the eyes. Foamy contents in the trachea was noted for groups, respectively; this finding was also noted for 1 pup in the control group. A short tail was noted for 1 pup each in the control and 50 mg/kg/day groups. In the 50 mg/kg/day group, 1 up was noted with yellow areas on the liver and 1 Pup was noted with a fractured tail. Dark red discoloration of the eyes was noted for 1 pup each in the control and 20 mg/kg/day groups; 11 pup was also noted with a small left eye. The aforementioned findings were noted insingle pups or litters, occurred infrequently or similarly in the control group, and/or did not occur in a dose-related manner, and therefore were not considered test substance-related.
- At the PND 21 necropsy of F1 weanlings selected for hormone analysis, no internal findings that could be attributed to parental administration of the test substance were noted at any dosage level. Internal findings were limited to dark red discoloration on the thyroid glands for a Male Pup in the 20 mg/kg/day group and an accessory spleen for a Female Pup in the 150 mg/kg/day group. Because these findings were limited to a single fetus and/or did not occur in a dose-related manner, they were not considered test substance-related.
- Thickened stomachs were observed in the 20, 50, and 150 mg/kg/day group F1 males and the 150 mg/kg/day group F1 females in Cohort 1A and were considered test substance-related. At the scheduled F1 male and female necropsies for Cohort 1B, thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females and were considered test substance-related; given the lack of systemic toxicity

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Test substance-related microscopic changes were observed in the nonglandular stomach for F1 males and females in Cohort 1A at all dosage levels. Hyperkeratosis was observed in all test substance-administered groups while epithelial hyperplasia was observed in the 50 and 150 mg/kg/day groups. The findings were not associated with clinical pathology changes but were of similar, although slightly less, severity when compared to the F0 generation and were considered adverse in the 150 mg/kg/day group males and females.
There were no test substance-related effects on ovarian primordial follicle counts in the 150 mg/kg/day group F1 females in Cohort 1A compared to the control group.

Other effects:

no effects observed

Description (incidence and severity):

- No test substance-related effects on anogenital distance were noted for F1 pups in the 20, 50, and 150 mg/kg/day groups. Longer anogenital distances (absolute and relative to the cube root of pup body weight) were noted for F1 male pups in the test substance-treated groups compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). However, the aforementioned differe ces did not occur in a dose-related manner and the mean absolute values in the 20, 50, and 150 mg/kg/day groups (4.07 mm, 4.19 mm, and 4.17 mm, respectively) were within the range of values in the Charles River Ashland historical.
- Areolae/nipple anlagen in the F1 male pups was not affected by test substance administration. There was no retention of nipples noted in any male pup on study on PND 12.
- There were no test substance-related changes in total T4 or TSH in pups that were culled on PND 4.
- There were no test substance-related changes in serum T4 or TSH in F1 weanlings on PND 21.
- There were no test substance-related changes in serum T4 or TSH in F1 animals assigned to Cohort 1A.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Dose descriptor:

LOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: local effects (nonglandular stomach)

Critical effects observed:

no

Reproductive effects observed:

no

Reference 6

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Anogenital distance, a triggered end point as per test guidelines, was not measured in the F2 pups because there were no significant effects observed on F1 sex ratio or age at vaginal opening or preputial separation.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

yes

Remarks:

See above

Qualifier:

according to guideline

Guideline:

other: JMAFF, Guideline 2-1-17, Reproduction Study (2000)

Deviations:

yes

Remarks:

See above

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test material name: Methyl acrylate
Chemical name: 2-Propenoic acid methyl ester
Synonyms: Acrylic acid methyl ester, Methyl-2-propenoate
Source: The Dow Chemical Company, Bayport, Texas
Reference: Lot: VF1301RFM1
Purity: 99.9%
Characterization: The purity of the test material was determined to be 99.9 ± 0.001% by area percent gas chromatography flame ionization detection, 300 ± 17 ppm water by Karl Fischer titration with identification by gas chromatography mass spectrometry and nuclear magnetic resonance (Binkley et al., 2007).
Appearance: Colorless liquid
Vapor Pressure: 68 mm Hg at 20°C
Odor: Acrid odor
Molecular Formula: C4H6O2
Molecular Weight: 86.1

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Laboratories Inc. (Portage, Michigan)
Age: Approximately six weeks at initiation of treatment.

Physical and Acclimation
Each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International). The animals were housed two-three per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study.

Housing
After assignment to study, animals were housed singly in stainless steel cages, except during breeding (one male and one female) and during the littering phases of the study. During littering, dams (and their litters) were housed in plastic cages provided with ground corncob nesting material from approximately GD 19 until completion of lactation. Cages had wire mesh floors and were suspended above catch pans. Non-woven gauze were placed in the cages to provide a cushion from the flooring for rodent feet and also provided environmental enrichment. In order to better visualize copulation and plugs, gauze was not placed in cages during the breeding phase. Cages contained a feed crock and a pressure activated lixit valve-type watering system. Environmental conditions were maintained as follows:
Temperature: 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
Humidity: 40-70%
Air Changes: 12-15 times/hour
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Randomization and Identification
Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Feed and Water
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water was provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. There were no contaminants in either the feed or the water that would have impacted the results of this study. Copies of these analyses are maintained at Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Animal Welfare
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities to be used for this study were DART 01 and Animal ID 01.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

Vapor Generating System
Chambers
The animals were exposed to filtered air or methyl acrylate vapors in 14.5 m3 inhalation exposure chambers under dynamic airflow conditions. Chamber airflow was maintained at approximately 2900 liters per minute, a rate that is sufficient to provide 12 air changes per hour and thus maintain normal concentrations of oxygen. The chambers were operated at a slight negative pressure relative to the surrounding area. All test chamber exhaust was passed through an activated charcoal bed to remove test material from the exhaust stream.

Prior to the start of the study, each of the chambers was checked to ensure that a uniform distribution of methyl acrylate vapor was present throughout the breathing zone of the animals.

Generation System
The various concentrations of methyl acrylate were generated using the glass J-tube method (Miller et al., 1980). Liquid test material was pumped into the glass J-tube assemblies (1 per exposure chamber) and vaporized by the flow of nitrogen gas passing through the bead bed of the glass J-tube. The nitrogen was heated as needed with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. All chambers, including the 0 ppm (control) chamber received the same amount (20 liters per minute) of supplemental nitrogen (carrier gas). The minimum amount of nitrogen necessary to reach the desired chamber concentrations was used. The generation system was electrically grounded and the J-tubes were changed as needed. The vaporized test material and carrier gas were mixed and diluted with supply air to achieve a total flow of approximately 2900 liters per minute at the desired test chamber concentration.

Exposure Environmental Conditions
Airflow through the chambers was determined with a manometer, which measures the pressure drop across a calibrated orifice plate, and was maintained at approximately 450 liters per minute. Chamber airflow data were collected using Setra Differential Pressure Transducers (Setra Systems, Inc., Boxborough, Massachusetts). The signal from the pressure transducer was sent to the CAMILE TG 4 Acquisition and Control System and recorded in liters per minute. The differential pressure transducer was calibrated with a gas meter (Singer Aluminum Diaphragm Meter, Model AL-2300, American Meter Division, Philadelphia, Pennsylvania) prior to the start of the study. Chamber temperature and relative humidity data were collected using Omega HX94C Probes (Omega Engineering Inc., Stamford, Connecticut) coupled to the CAMILE TG 4 Data Acquisition and Control System. The chamber temperature and relative humidity were controlled by a system designed to maintain values of approximately 22 ± 2°C and 40 to 60%, respectively. Chamber temperature, relative humidity, and airflow data were manually recorded from the CAMILE TG 4 Data Acquisition and Control System once per hour.

Chamber Environmental Conditions
Chamber temperatures values for all chambers were maintained between 20.3 and 23.3°C. Chamber relative humidity was maintained in the range of 35.9 and 64.5% for all exposure chambers. Minor excursions of daily values from the protocol-specified relative humidity range (40-60%) for the chambers were noted. These minor excursions did not affect the integrity of the study. Chamber airflow in all four chambers was maintained throughout
the study duration, ensuring 12-15 calculated air changes per hour at approximately 2900 liters per minute of total airflow.

Details on mating procedure:

Breeding for the P1 and P2 adults commenced after approximately ten weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until mating occurred or two weeks elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then removed from the males and returned to their home cages. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating. If available, one rat/sex/litter was randomly selected for the P2 mating to produce the F2 generation. More than one weanling may have been selected from the litters, if necessary, to achieve 27 breeding pairs/dose level for the second generation. Cohabitation of P2 male and female littermates was avoided. In cases where a mating partner had died or was otherwise not available, the animal was paired with the next available partner. A second breeding of the first or second generation adults was not conducted.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chamber concentrations of methyl acrylate, measured approximately in the center of the breathing zone of the animals, were determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer (Foxboro/Wilks, South Norwalk, Connecticut) and reported by a strip chart recorder. The IR spectrophotometer was calibrated and a standard curve was compiled prior to and at the midpoint of the study, using air standards prepared by vaporizing measured volumes of methyl acrylate into Tedlar sample bags (Series 233, SKC, Eighty Four, Pennsylvania) along with the metered volumes of dry, compressed air. Chamber concentrations during the exposures were interpolated using the standard curve. The analytical system was checked prior to each exposure with a methyl acrylate standard gasbag of known concentration. The CAMILE TG 4 Data Acquisition and Control System toggled the IR spectrophotometer between the chambers for concentration sampling. The nominal concentration of the test material in each chamber was estimated based on the amount of test material used and the total airflow through the chamber. Prior to the start of the study, each of the chambers was checked to ensure that a uniform distribution of vapor was present throughout the breathing zone of the animals.

Duration of treatment / exposure:

Six hours/day, seven days/week for approximately 10 weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (four weeks) for each of two generations

Frequency of treatment:

Daily. Maternal rats were not exposed to methyl acrylate after GD 20 through LD 4 in order to allow for parturition and initiation of lactation.

Details on study schedule:

Groups of 27 male and 27 female Crl:CD(SD) (Sprague-Dawley derived) were exposed to targeted concentrations of 0, 5, 25, or 75 ppm methyl acrylate for six hours/day, seven days/week for approximately 10 weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (four weeks) for each of two generations. Maternal rats were not exposed to methyl acrylate after GD 20 through LD 4 in order to allow for parturition and initiation of lactation. Females with no evidence of mating were exposed until termination. Maternal rats were exposed from LD 5-LD 28. During the lactation period, pups were not placed in the exposure chambers, but remained in nesting boxes separated from the dam for approximately six hours /day on PND 5-28. Due to the daily exposures of the dams and separation from the pups, weaning occurred on PND 28 to allow the pups extra time to grow prior to single housing. Weanlings selected for the second generation began exposure on PND 28. Weanlings that were not selected for the second generation were not placed in the exposure chambers on PND 28, but were necropsied on PND 29. A comprehensive evaluation of male and female reproductive systems was conducted, and included an evaluation of gonadal function, the estrous cycle, mating performance, conception, gestation, parturition and lactation, as well as survival, growth and development of the offspring. In-life observations, body weights, feed consumption and litter data were evaluated. In addition, a gross necropsy of the P1 and P2 adults was conducted with extensive histopathologic examination of reproductive organs and target tissues. The material administration began on April 18, 2008 and was continued up until the day prior to necropsy. The F1weanlings were necropsied from August 18-31, 2008. The P1 adults were necropsied from August 11-14, 2008 (males) and September 3-4, 2008 (females). The F2 weanlings were necropsied from December 29, 2008 to January 11, 2009. The P2 adults were necropsied from January 5-8, 2009 (males) and January 12-13, 2009 (females).

Dose / conc.:

5.3 ppm (nominal)

Remarks:

5.3 ± 0.2 ppm

Dose / conc.:

24.9 ppm (nominal)

Remarks:

24.9 ± 0.4 ppm

Dose / conc.:

73.4 ppm (nominal)

Remarks:

73.4 ± 1.8 ppm

Dose / conc.:

5.3 ppm (analytical)

Remarks:

5.3 ± 0.2 ppm (corresponding to approx. 0.019 mg/L)

Dose / conc.:

25.7 ppm (analytical)

Remarks:

25.7 ± 0.3 ppm (corresponding to approx. 0.092 mg/L)

Dose / conc.:

75.4 ppm (analytical)

Remarks:

75.4 ± 0.6 ppm (corresponding to approx. 0.269 mg/L)

No. of animals per sex per dose:

27/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

In-life parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In addition, post-mortem evaluations included gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.

Positive control:

None

Parental animals: Observations and examinations:

Daily Observations
A cage-side examination was conducted twice daily, approximately the same time eachday. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could be observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Any animals found dead were necropsied as soon as practical. In addition, all animals and litters if available were
observed for morbidity, mortality, and the availability of feed and water at least twice daily. Moribund animals not expected to survive until the next observation period were humanely euthanized that day. In addition, females were observed for signs of parturition beginning on or about GD 20.

Clinical Observations
Clinical observations were performed immediately after exposure, with the exception of day 1, which was conducted pre-exposure. Clinical examinations were conducted weekly on all males throughout the study, and weekly on all females throughout the pre-breeding period. Mated (sperm-positive or plug-positive) females received clinical examinations on GD 0, 7, 14 and 21. Females were observed for signs of parturition beginning on or about GD 20. Dams unable to deliver or exhibiting signs of severe dystocia were humanely euthanized and necropsied. Females that delivered litters were subsequently evaluated on LD 0, 1, 4, 7, 14, 21 and 28. Clinical observations were not conducted on females that failed to mate or deliver a litter for the remainder of the study until the week prior to the scheduled necropsy, unless deemed appropriate based on cageside observations. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

Body Weights/Body Weight Gains
All rats were weighed during the pre-exposure period and weekly during the 10-week prebreeding periods. Males continued to be weighed weekly after the pre-breeding period until termination. Mated females were weighed on GD 0, 7, 14, and 21. Lactating females were weighed on LD 1, 4, 7, 14, 21 and 28. Females that failed to mate and/or deliver a litter were weighed during the subsequent gestation and/or lactation segments of the study. Body weight gains of females were calculated for the following intervals in both generations: GD 0-7, 7-14, 14-21, 0-21 and LD 1-4, 4-7, 7-14, 14-21, 21-28 and 1-28.

Feed Consumption
Feed consumption was determined weekly during the 10-week pre-breeding period for all animals by weighing feed containers at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption was measured weekly in males and dietary concentrations were adjusted accordingly. During gestation, feed consumption was measured at weekly intervals of sperm-/or plug- positive females on GD 0, 7, 14, and 21. Feed consumption was not recorded for non-confirmed mated females. During lactation, feed consumption was measured on LD 1, 4, 7, 11, 14, 17, 19, 21, 23, 26, and 28. Feed consumption was not measured in females that failed to mate or deliver a litter.

Oestrous cyclicity (parental animals):

Vaginal lavage samples were collected daily for all P1 and P2 females for three weeks prior to mating and during cohabitation until each female was sperm or plug positive or until the two-week mating period elapsed. Lavage samples were collected by gently irrigating the vagina with water and transferring loosely adherent vaginal cells to a slide with a pipette. Vaginal lavage slides were examined to determine estrous cycle length and pattern. Additionally, on the day of scheduled necropsy, the stage within the estrous cycle was determined for all P1 and P2 female rats.

Sperm parameters (parental animals):

Sperm parameters were evaluated in all P1 and P1 males at termination. Unless circumstances dictated otherwise, the left and right epididymides and testes were allocated as follows: right epididymis – motility and histopathology; left epididymis – counts; right testis – histopathology; left testis – counts.

Motility
Immediately after euthanasia of males and isolation of their epididymides, a small sample of sperm from the right cauda epididymis was expressed into a dish containing SpermPrep Medium (ZDL, Inc., Lexington, Kentucky) and was incubated at room temperature for approximately 2-3 minutes. An aliquot of the incubated sperm suspension was placed in a chamber of the HTM Integrated Visual Optical System (IVOS; Hamilton-Thorne Research, Beverly, Massachusetts) for the determination of total percent motile (showing any motion) and percent progressively motile (showing net forward motion) sperm. Images from the motility analyses were recorded on CD-R and archived with the study file. After sperm were released, the epididymis was placed in Bouin’s fixative and saved for histological examination.

Counts
The left testis and cauda epididymis were weighed and frozen at -20°C for subsequent determination of the number of homogenization-resistant spermatids and sperm per testis/cauda epididymis and per gram of testicular/epididymal tissue. The thawed testis or epididymis were minced, diluted and stained with a fluorescent DNA-binding dye (HTM-IDENT, Hamilton-Thorne Research, Beverly, Massachusetts) and the spermatid or sperm count was determined from an aliquot loaded into the IVOS analyzer as described by Stradler et al. (1996). Because there were no treatment-related differences in testicular/epididymal sperm counts, only samples from the high-dose and control animals were evaluated.

Morphology
An aliquot of sperm suspension was placed on a slide, and a smear was prepared and then air dried for subsequent evaluation of sperm morphology. At least 200 sperm from each control and high-dose group male were evaluated and classified as normal or abnormal as described by Filler (1993). Sperm morphology was scored blind with respect to treatment group.

Litter observations:

Litter Observations
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. The following information was recorded for each litter: the date of parturition, the number of live and dead pups on LD 0, 1, 4, 7, 14, 21 and 28, and the sex and body weight of each pup on LD 1, 4 (before and after culling), 7, 14, 21 and 28. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. In addition, pup clinical observations were recorded on each litter on PND 0, 1, 4, 7, 14, 21 and 28. Any pups found dead or sacrificed in moribund condition were sexed and examined grossly if possible for external and visceral defects. These pups were preserved in neutral, phosphate-buffered 10% formalin.

Culling
To minimize variation in pup growth due to differences in litter size, F1 and F2 litters were standardized to eight/litter on PND 4. This was accomplished by randomly ordering the pups in each litter by sex. Pups to be culled were then randomly selected using a computer generated selection procedure, so that four males and four females (whenever possible) remained in each litter. Litters with eight or fewer pups were not culled. Culled pups were euthanized by administration of Socumb euthanasia solution (Veterinary Laboratories, Inc., Lenexa, Kansas) into the buccal cavity, and then discarded.

Postmortem examinations (parental animals):

Necropsy
Adult males (fasted) were submitted for necropsy after completion of their respective mating period when it was deemed that they were no longer needed for assessment of reproductive effects. Adult females (fasted) were terminated after weaning of their litters, or at least 24 days after the end of the mating period for females not producing a litter. On the morning of the scheduled necropsy, all surviving P1 and P2 males and females were weighed. Vaginal lavage smears were prepared from all surviving P1 and P2 females for later determination of estrous cycle stage. The animals were anesthetized by the inhalation of CO2, the tracheas were exposed and clamped, and the animals were euthanized by decapitation.

A complete necropsy was conducted on all animals by a veterinary pathologist or a technician qualified to recognize lesions, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.

The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain (Kopf et al., 1964) for approximately two minutes and were examined for the presence and number of implantation sites. After evaluation, uteri was gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.

Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, brain, pituitary (weighed after fixation), liver, kidneys, adrenal glands, spleen, thyroid with parathyroids (weighed after fixation) were recorded, and the organ-to-body weight ratios calculated. In addition, weights of the left testis and left cauda epididymis were collected for use in calculating sperm count parameters.

Representative samples of tissues listed in Table 3 were collected and preserved in neutral, phosphate-buffered 10% formalin, except that the right testis, right epididymis, and ovaries (P2 females only) was preserved in Bouin’s or another appropriate fixative. Transponders were removed and placed in jars with the tissues. During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Similar necropsy procedures were followed for these animals except that terminal body and organ weights were not recorded and the testes, epididymides and ovaries were preserved in neutral, phosphate-buffered 10% formalin.

Histopathology
Histologic examination of the tissues was conducted on all control and high-dose adult rats, and on the reproductive organs of any study rats with
signs of reduced fertility. The thyroid glands of all P2 adult male and female control and high-dose rats were examined microscopically because the absolute thyroid gland weights of P2 high-dose males and females were statistically identified as lower than controls. Examination of additional tissues from the low- and mid-dose groups was limited to the nasal tissues/pharynx and relevant gross lesions. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope.

Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at 5 μm and stained with modified periodic acid Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis were qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).

Examination of the ovaries included enumeration of primordial follicles using a method similar to Bucci et al. (1997). From among the surviving post-lactational P2 females in the control and high-dose groups, 15 per group were randomly selected for this examination.

Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was neither expected to
significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue as adversely affected, but not to the point of organ failure. The health status of the animal may or may not be affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not life threatening. A grade of severe was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may be life threatening.

A complete set of tissues was examined from rats found dead, moribund, or euthanized due to accidental trauma. Histological examination was
conducted in a similar manner as described above, except that the testes were stained with hematoxylin and eosin.

Postmortem examinations (offspring):

Necropsy
Three pups/sex/litter from the F1 and F2 litters randomly selected at the time of weaning were submitted on PND 29 for a complete necropsy by a veterinary pathologist or a technician qualified to recognize lesions, assisted by a team of trained individuals. Pups were anesthetized with CO2, weighed and euthanized by decapitation. Gross pathological examination was performed as described above for adults, except that the weanlings were not fasted overnight. Representative sample of grossly abnormal tissues and any known target organs were collected from all weanlings at the scheduled necropsy. In addition, one of the three pups/sex/litter was randomly selected from those examined grossly for the collection of brain, spleen, uterus, and thymus weights. Organ-to-body weight ratios were calculated. The brain, spleen, thymus, gross lesions and known target organs were preserved in neutral, phosphate-buffered 10% formalin. Dead or moribund pups were examined in a similar manner for possible defects and/or cause of death and were preserved in neutral, phosphate-buffered 10% formalin.

Statistics:

See below "Any other information on materials and methods incl. tables".

Reproductive indices:

Reproductive indices were calculated for all dose level groups as follows:
• Female mating index = (No. females with evidence of mating/No. paired) x 100
• Male mating index = (No. males with evidence of mating/No. paired) x 100
• Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
• Male conception index = (No. males siring a litter/No. mated) x 100
• Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
• Male fertility index = (No. males siring a litter/No. paired) x 100
• Gestation index = (No. females delivering a viable litter/No. females with evidence of pregnancy) x 100
• Gestation survival index = percentage of delivered pups alive at birth
• Post-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100
• Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100
• Day 7, 14, 21 or 28 pup survival index = (No. viable pups on day 7, 14, 21 or 28/No. live after culling) x 100

Offspring viability indices:

• Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100
• Day 7, 14, 21 or 28 pup survival index = (No. viable pups on day 7, 14, 21 or 28/No. live after culling) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on behavior or demeanor were observed in any phase of the study at any dose level. A number of incidental observations bearing no relation to treatment were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One P1 male exposed to 75 ppm died on test day 70. The cause of death was not determined. One P1 male exposed to 75 ppm was euthanized moribund on test day 106. The cause of moribundity was urolithiasis, with associated inflammation and transitional cell hyperplasia of the urinary bladder and kidneys. One P1 male from the control group was euthanized moribund on test day 98. The cause of moribundity was lymphoid cell leukemia. Another P1 male from the control was euthanized on test day 113 due to accidental fracture of the upper jaw.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a treatment-related decrease in the body weight of P1 males of the 75 ppm exposure group when compared to controls, although these differences only reached statistical significance on TD 7 and 14 throughout the entire first generation. Similarly, there was a treatment-related decrease in the body weight of P1 females of the 75 ppm exposure group, which reached statistical significance on three days (TD 21, 63, and 70) of the 10-week premating period. There was a treatment-related decrease in the body weight of P1 females of the 75 ppm exposure group across the entire
gestation and lactation period. However, gestation body weight gain of the 75 ppm females remained comparable to controls. During lactation, the 75 ppm females did not lose as much body weight as controls, which could have been related to their lower body weight at the start of lactation. There were no treatment-related differences in body weight of P1 males and body weight/body weight gain of P1 females exposed to 25 or 5 ppm methyl acrylate when compared to controls. The difference in body weight of 5 ppm males (TD 7) and gestation body weight gain of 5 ppm females (GD 0-7) was statistically identified and decreased when compared to their respective controls. However, this was not considered to be a treatmentrelated effect due to the lack of a dose-response relationship and the low incidence.

Selected P1 Male Body Weights
Mean Body Weight (g)
ppm 0 5 25 75
TD 1 193.9 190.4 193.6 191.9
TD 7 237.8 228.0* 235.9 226.3*
TD 14 291.1 286.8 284.9 276.7*
TD 28 369.3 358.5 366.7 358.6
TD 70 512.4 492.5 499.2 487.3
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Selected P1 Female Pre-Breeding Body Weights
Mean Body Weight (g)
ppm 0 5 25 75
TD 1 139.4 136.4 138.9 139.8
TD 7 158.7 153.8 159.0 152.5
TD 21 203.8 203.0 204.6 191.5*
TD 63 273.1 266.1 272.4 248.1*
TD 70 281.7 272.0 276.6 258.5*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05

Selected P1 Gestation/Lactation Body Weights/Body Weight Gains
Gestation Mean Body Weight (g)
ppm 0 5 25 75
GD 0 287.4 280.1 284.3 261.8*
GD 7 319.3 302.7 315.8 289.0*
GD 14 350.1 337.2 347.5 318.5*
GD 21 433.8 429.4 435.9 404.6*

Gestation Mean Body Weight Gains (g)
GD 0-21 146.4 149.3 151.6 142.9
Lactation Mean Body Weight (g)
LD 1 330.9 316.4 321.6 297.7*
LD 4 343.8 335.6 337.2 312.6*
LD 7 328.0 323.0 324.4 297.9*
LD 14 336.9 334.8 335.6 308.8*
LD 28 313.4 306.6 309.3 294.6*
Lactation Mean Body Weight Gains (g)
LD 1-28 -17.5 -9.8 -12.3 -3.1*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a treatment-related decrease in feed consumption of the P1 males in the 75 ppm exposure group when compared to controls, although these differences only reached statistical significance for three measurement intervals (TD 1-7, 7-14, and 56-63) throughout the first generation. During the 10-week premating period, there was a treatment-related decrease in feed consumption of the P1 females in the 75 ppm exposure group when compared to controls, and these differences reached statistical significance for most measurement intervals. Feed consumption of the 75 ppm females was also decreased throughout gestation (≤ 12%) when compared to controls. During lactation, feed consumption of the 75 ppm females was slightly decreased when compared to controls, although these differences only reached statistical significance for three measurement intervals (LD 14-17, 17-19, and 23-26). There were no treatment-related or statistical differences in feed consumption of P1 males and females exposed to 25 or 5 ppm methyl acrylate when compared to controls.

Selected P1 Male Feed Consumption Data
Mean g/Animal/Day
ppm: 0 5 25 75
TD 1-7 22.4 21.7 22.8 20.0*
TD 7-14 24.2 24.8 24.7 23.0*
TD 21-28 24.9 24.1 25.9 24.8
TD 42-49 26.8 26.1 26.1 25.7
TD 56-63 27.4 26.0 26.6 25.0*
TD 86-91 27.1 25.8 27.4 25.5
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Selected P1 Female Feed Consumption Data
Mean g/Animal/Day
ppm: 0 5 25 75
Premating days 1-7 16.2 15.0 15.9 14.2$
Premating days 7-14 16.6 16.9 16.6 15.4*
Premating days 21-28 17.6 17.3 17.7 16.7
Premating days 42-49 18.8 18.0 17.8 17.3*
Premating days 56-63 18.0 17.2 17.2 13.3$
Premating days 63-70 18.1 17.4 17.3 18.8
GD 0-7 22.6 21.3 22.1 19.9*
GD 7-14 23.8 22.9 23.8 21.7*
GD 14-21 23.2 23.1 22.6 21.0*
LD 1-4 29.4 32.1 29.8 30.4
LD 7-11 40.6 41.8 41.2 38.1
LD 14-17 51.8 52.1 50.6 48.3$
LD 23-26 90.9 90.9 92.5 87.3$
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.
$ Statistically different from control mean by Wilcoxon’s test, alpha = 0.05.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related histopathologic effects were present in the nasal tissues of P1 and P2 males and females given 25 or 75 ppm. The incidence and severity of the nasal effects were dose-related. Degeneration with regeneration of the olfactory epithelium (very slight to moderate) occurred in all P1 and P2 males and females exposed to 75 ppm. The degeneration consisted of thinning and disarray of the olfactory epithelial cells, which was most prevalent in the anterior and dorsal aspects of the nasal passages. Regenerative hyperplasia of the olfactory epithelium accompanied the degenerative change in multifocal sites. A lesser degree of multifocal olfactory epithelial degeneration (very slight), without accompanying regenerative hyperplasia, was noted in 7/27 P1 females exposed to 25 ppm, and in 6/27 P2 males and 8/27 P2 females exposed to 25 ppm. One P1 female and one P2 female exposed to 5 ppm also had very slight multifocal olfactory epithelial degeneration. However, the degeneration was located in only two sites of the nose for both of these animals, and therefore was interpreted to be comparable with spontaneous focal olfactory epithelial degeneration that was noted in 2/27 control group P1 females and 3/27 control group P2 females, and not an effect of treatment.

There were several histopathologic effects that accompanied the degeneration of the olfactory epithelium. Very slight or slight degeneration of the olfactory nerve was present in most of the P1 and P2 males and females exposed to 75 ppm, and one P1 male exposed to 25 ppm. This effect was characterized by thinning of the axons and reduction in the diameter of the olfactory nerve fascicles in areas of olfactory epithelial degeneration. Very slight or slight multifocal chronic-active inflammation accompanied the olfactory epithelial degeneration in 16/27 P1 males, 20/27 P1 females, 14/27 P2 males, and 8/27 P2 females exposed to 75 ppm, and in one or two males and females from both generations exposed to 25 ppm. The inflammation
consisted of neutrophils in the olfactory epithelium, with or without the presence of a mucopurulent exudate. Very slight multifocal necrosis of individual olfactory epithelial cells, with or without exfoliation of necrotic cells into the lumen of the nasal passages, was present in most of the P1 and P2 males and females exposed to 75 ppm, and a few P1 and P2 animals (one to four per sex) exposed to 25 ppm. One P1 female exposed to 5 ppm also had very slight multifocal necrosis of individual olfactory epithelial cells. However, the necrosis was located in only two sites of the nose in this animal, and therefore was interpreted to be comparable with spontaneous focal olfactory epithelial cell necrosis that was noted in one control group P1 male, one control group P1 female, and one control group P2 female, and not an effect of treatment.

A treatment-related increase in the incidence of very slight or slight multifocal hyperplasia of the transitional epithelium that covers the nasal turbinates was present in P1 and P2 males and females exposed to 25 or 75 ppm. The incidence and severity of transitional epithelial hyperplasia in P1 and P2 males and females exposed to 5 ppm was comparable to controls.

A treatment-related increase in the incidence of very slight or slight diffuse hyperplasia and hypertrophy of the respiratory epithelium that covers the nasal septum and dorsal portion of the anterior nasal cavity was present in P1 males and females exposed to 25 or 75 ppm, and in P2 males and females exposed to 75 ppm. The incidence and severity of respiratory epithelial hyperplasia and hypertrophy in P1 and P2 males and females exposed to 5 ppm was comparable to controls.

Treatment-related very slight focal or multifocal mineralization of the olfactory epithelium was present in one or two P1 and P2 animals exposed to 25 ppm, 6/27 P1 males, 4/27 P1 females, 16/27 P2 males and 14/27 P2 females exposed to 75 ppm. The mineralization was present in areas olfactory epithelial degeneration. One P1 female exposed to 75 ppm also had slight multifocal mineralization of the nasal respiratory epithelium that was interpreted to be treatment related. Other treatmentrelated nasal effects consisted of very slight multifocal squamous metaplasia of the transitional epithelium in 5/27 P1 males exposed to 75 ppm, and ulceration of the olfactory epithelium in four P1 males, one P1 female, and one P2 female exposed to
75 ppm.

All other histopathologic observations were considered to be spontaneous alterations, or caused by accidental trauma, unassociated with inhalation exposure of methyl acrylate. There were no histopathologic systemic effects in P1 or P2 rats at any exposure level. The NOEC for histopathologic nasal effects was 5 ppm.


Histopathologic Nasal Tissue Effects – P1 Males
Dose (ppm) 0 5 25 75
NASAL TISSUE - PHARYNX (number examined) (27) (27) (27) (27)
Degeneration, olfactory nerve, multifocal -very slight 0 0 1a 11a
-slight 0 0 0 14a
Degeneration with Regeneration, olfactory epithelium, multifocal
-slight 0 0 1a 12a
-moderate 0 0 0 15a
Hyperplasia, transitional epithelium; multifocal -very slight 3 4 17a 12a
-slight 0 0 0 15a
Hyperplasia and Hypertrophy, goblet cell, respiratory epithelium, diffuse
-very slight 1 0 4a 1
-slight 0 0 1a 22a
Inflammation, chronic active, olfactory epithelium, multifocal
-very slight 1 0 2 15a
-slight 0 0 0 1a
Metaplasia, squamous, transitional epithelium, multifocal -very slight 0 0 0 5a
Mineralization, olfactory epithelium, focal -very slight 0 0 0 1a
Mineralization, olfactory epithelium, multifocal -very slight 0 0 1a 5a
Necrosis, individual cell, olfactory epithelium, focal -very slight 1 0 1 0
Necrosis, individual cell, olfactory epithelium, multifocal -very slight 0 0 1a 26a
Ulcer, olfactory epithelium, focal -very slight 0 0 0 4a
a- Indicates the effects judged to be treatment-related.

Histopathologic Nasal Tissue Effects
P1 Females
Dose (ppm) 0 5 25 75
NASAL TISSUE - PHARYNX (number examined) (27) (27) (27) (27)
Degeneration, olfactory epithelium, focal -very slight 2 1 4 0
Degeneration, olfactory epithelium, multifocal -very slight 0 1 7a 0
Degeneration, olfactory nerve, multifocal -very slight 0 0 0 8a
-slight 0 0 0 19a
Degeneration with Regeneration, olfactory epithelium, multifocal-slight 0 0 0 8a
-moderate 0 0 0 19a
Hyperplasia, transitional epithelium; multifocal -very slight 2 1 23a 24a
-slight 0 0 2a 3a
Hyperplasia and Hypertrophy, goblet cell, respiratory epithelium, diffuse-very slight 0 1 8a 10a
-slight 0 0 13a 1a
Inflammation, chronic active, olfactory epithelium, multifocal-very slight 0 0 1 20a
Inflammation, chronic active, respiratory epithelium, multifocal-very slight 0 0 1 5a
Mineralization, olfactory epithelium, focal -very slight 0 0 0 2a
Mineralization, olfactory epithelium, multifocal -very slight 0 0 0 2a
Mineralization, respiratory epithelium, focal -slight 0 0 0 1a
Necrosis, individual cell, olfactory epithelium, focal -very slight 1 0 3 0
Necrosis, individual cell, olfactory epithelium, multifocal -very slight 0 1 2a 26a
Ulcer, olfactory epithelium, focal -very slight 0 0 0 1a
a-Indicates the effects judged to be treatment-related.

Histologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effects of treatment. There were no treatment-related or statistically-identified differences in the mean number of small and growing ovarian follicles in females exposed to 75 ppm as compared to females from the control group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no evidence of an effect on estrous cyclicity at any dose level of methyl acrylate in either generation.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no treatment-related effects of methyl acrylate on any sperm analysis parameter at any exposure level in either generation. There was a statistically identified increase in epididymal and testicular sperm counts of P1 males of the 75 ppm exposure group when compared to controls, which was due to two males in the control group (1258 and 1263) with very low sperm counts.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects of treatment at any exposure level on mating, conception, fertility or gestation indices, post-implantation loss, time to mating or gestation length in either generation.

Dose descriptor:

NOEC

Remarks:

reproductive toxicity

Effect level:

ca. 0.269 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 75 ppm; the highest concentration tested.

Dose descriptor:

NOEC

Remarks:

parental local toxicity

Effect level:

ca. 0.019 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 5 ppm; based on histologic changes in nasal tissues.

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on behavior or demeanor were observed in any phase of the study at any dose level. A number of incidental observations bearing no relation to treatment were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One P2 female exposed to 25 ppm (animal number 08A5190) was euthanized on test day 57 due to severe inflammation of the hind feet. One P2 male exposed to 5 ppm (animal number 08A5040) was euthanized moribund on test day 87. The cause of moribundity was severe inflammation of the periodontal tissue associated with fracture of the upper incisors. One P2 female from the control group (animal number 08A5134) was euthanized on test day 70 due to accidental fracture of the nose.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a treatment-related decrease in the body weight of P2 males of the 75 ppm exposure group when compared to controls, which was statistically identified throughout the entire second generation. Similarly, there was a treatment-related decrease in the body weight of P2 females of the 75 ppm exposure group, which reached statistical significance on all measurement days during the 10-week premating period. There was a treatment-related decrease in the body weight of P2 females of the 75 ppm exposure group across the entire gestation and lactation periods. The gestation body weight gains of the 75 ppm females were significantly lower than control values, however, this difference was equivocal when all dose groups were considered. As in the P1 females, the net body weight loss typical of lactating female rats was less in the 75 ppm females than it was in controls. There were no treatment-related differences in body weight of P2 males and body weight/body weight gain of P2 females exposed to 25 or 5 ppm methyl acrylate when compared to controls. Statistical differences in the body weight/body weight gain of 25 or 5 ppm females were not considered to be treatment-related due to the lack of a doseresponse relationship and/or the low incidence.

Selected P2 Male Body Weights
Mean Body Weight (g)
ppm 0 5 25 75
TD 1 169.7 158.0 170.9 148.4*
TD 6 212.6 200.7 213.6 181.2*
TD 13 274.6 258.9 277.9 239.6*
TD 27 373.7 365.2 379.0 330.4*
TD 69 538.4 512.4 540.3 472.3*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Selected P2 Female Pre-Breeding Body Weights
Mean Body Weight (g)
ppm 0 5 25 75
TD 1 143.6 135.6 135.6 125.1*
TD 6 166.4 160.0 156.4 144.3*
TD 13 194.3 187.0 183.1 169.9*
TD 27 238.9 229.4 228.3 206.4*
TD 69 299.5 288.1 293.2 264.1*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05
Selected P2 Gestation/Lactation Body Weights/Body Weight Gains
Gestation Mean Body Weight (g)
ppm 0 5 25 75
GD 0 298.7 292.5 290.2 266.5*
GD 7 334.0 319.7 321.7 294.7*
GD 14 367.1 350.4 353.3 324.4*
GD 21 459.3 437.6 440.5 412.4*
Gestation Mean Body Weight Gains (g)
GD 0-21 160.6 145.1* 150.3 145.9*
Lactation Mean Body Weight (g)
LD 1 345.1 328.3 337.0 303.8*
LD 4 365.4 345.6* 350.4 323.0*
LD 7 352.6 333.7 336.4 311.2*
LD 14 358.3 338.3* 341.1 321.2*
LD 28 322.5 310.0 314.4 298.0*
Lactation Mean Body Weight Gains (g)
LD 1-28 -22.6 -18.2 -22.5 -6.1*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a treatment-related decrease in feed consumption of the P2 males in the 75 ppm exposure group when compared to controls, and these differences reached statistical significance for the majority of measurement intervals throughout the second generation. During the 10-week premating period, there was a treatment-related decrease in feed consumption of the P2 females in the 75 ppm exposure group when compared to controls, and these differences also reached statistical significance for most measurement intervals. Feed consumption of the 75 ppm females was also decreased throughout gestation (≤ 10%) when compared to controls. During lactation, feed consumption of the 75 ppm females was slightly decreased when compared to controls, although these differences only reached statistical significance for two measurement intervals (LD 7-11 and 14-17). There were no treatment-related differences in feed consumption of P2 males and females exposed to 25 or 5 ppm methyl acrylate when compared to controls. The difference in feed consumption of 5 ppm males when compared to controls was statistically identified and decreased for two measurement intervals (TD 1-6 and 34-41), however, this was not considered to be a treatment-related effect due to the lack of both a dose-response relationship and temporal association.

Selected P2 Male Feed Consumption Data
Mean g/Animal/Day
ppm: 0 5 25 75
TD 1-6 24.3 22.6* 24.7 20.8*
TD 6-13 27.0 25.6 27.2 23.8*
TD 20-27 28.7 27.6 29.4 26.2*
TD 34-41 29.4 27.6* 29.5 26.9*
TD 41-48 28.7 27.6 29.9 27.0
TD 55-62 29.3 28.0 29.7 27.1*
TD 90-97 29.0 28.5 29.2 26.6*
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Selected P2 Female Feed Consumption Data
Mean g/Animal/Day
ppm: 0 5 25 75
Premating days 1-6 19.3 19.0 18.4 17.1$
Premating days 6-13 19.9 19.6 19.2 17.7*
Premating days 20-27 20.2 19.4 19.8 18.0*
Premating days 41-48 20.4 19.1 20.0 19.2
Premating days 55-62 18.6 19.1 18.9 18.8
Premating days 62-69 19.1 18.3 18.8 17.6*
GD 0-7 23.1 22.3 22.7 21.1*
GD 7-14 25.2 23.9 25.0 22.7*
GD 14-21 24.1 23.0 23.1 21.9*
LD 1-4 33.7 32.4 30.6 33.0
LD 7-11 46.0 44.7 43.7 41.9*
LD 14-17 55.3 53.7 53.0 51.1*
LD 23-26 95.0 93.2 92.5 91.9
* Statistically different from control mean by Dunnett’s test, alpha = 0.05.
$ Statistically different from control mean by Wilcoxon’s test, alpha = 0.05.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

P2 males and females exposed to 75 ppm had statistically identified treatment-related lower final body weights. The final body weights of P2 males and females exposed to 75 ppm were 13.1% and 9.6% lower than controls, respectively. The relative weights of the brain, testes, seminal vesicles (with coagulating glands) and epididymides of males exposed to 75 ppm, and the relative weights of the adrenals and brain of females exposed to 75 ppm were statistically identified as higher than controls. The absolute weights of the adrenals, kidneys, spleen, pituitary gland, and thyroid gland of males exposed to 75 ppm, and the absolute weights of the kidneys, spleen and thyroid gland of females exposed to 75 ppm were statistically identified as lower than controls. The organ weight alterations of males and females exposed to 75 ppm were interpreted to be reflective of the lower body weights of these animals. Males exposed to 25 ppm had statistically identified lower absolute and relative pituitary weights, and females exposed to 25 ppm had a statistically identified lower absolute thyroid weight. Males exposed to 5 ppm had a statistically identified higher relative testes weight, and females exposed to 5 ppm had statistically identified higher absolute and relative adrenal weights. The organ weight alterations from the 5 and 25 ppm dose groups were interpreted to be unrelated to treatment due to the lack of a clear dose response and/or the values were within historical controls
ranges of studies recently conducted at this laboratory.

Organ Weight Data – P2 Adults
Concentration (ppm)
0 Historical1 5 25 75
Parameter MALES
Final Body Weight (g) 624.4 606.9-674.8 599.9 627.6 542.9*a
Absolute Adrenal Glands (g) 0.055 0.062-0.073 0.054 0.056 0.048*
Absolute Kidneys (g) 4.058 3.924-4.350 4.010 4.002 3.671*
Relative Brain (g/100g bw 0.346 0.333-0.386 0.363 0.344 0.386*
Absolute Spleen (g) 0.895 0.845-0.963 0.905 0.867 0.783*
Relative Testes (g/100g bw) 0.582 0.571-0.641 0.636* 0.593 0.662*
Relative Seminal Vesicle (g/100g bw) 0.319 0.266-0.319 0.322 0.312 0.367*
Relative Epididymides (g/100g bw) 0.223 0.224-0.248 0.238 0.229 0.255*
Absolute Pituitary (g) 0.0149 0.009-0.017 0.0141 0.0137* 0.0134*
Relative Pituitary (g/100g bw) 0.0024 0.002-0.003 0.0024 0.0022* 0.0025
Absolute Thyroid Gland (g) 0.0253 0.0224-0.0284 0.0255 0.0269 0.0224*
Parameter FEMALES
Final Body Weight (g) 326.3 296.3-347.2 313.7 318.4 295.0*a
Absolute Adrenal Glands (g) 0.062 0.070-0.111 0.068* 0.064 0.067
Relative Adrenal Glands (g/100g bw) 0.019 0.023-0.035 0.022* 0.020 0.023*
Absolute Kidneys (g) 2.274 2.187-2.424 2.259 2.149 2.108*
Relative Brain (g/100g bw) 0.620 0.588-0.703 0.644 0.628 0.669*
Absolute Spleen (g) 0.585 0.593-0.620 0.543 0.562 0.513*
Absolute Thyroid Gland (g) 0.0203 0.0166-0.0202 0.0182 0.0180* 0.0180*
*Statistically different from control mean by Dunnett’s Test, alpha = 0.05.
1Historical controls group mean range from seven dietary studies reported between 2002 and 2006.
a-Values interpreted to be treatment-related effects.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be incidental findings, unassociated with exposure to methyl acrylate.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related histopathologic effects were present in the nasal tissues of P1 and P2 males and females given 25 or 75 ppm. The incidence and severity of the nasal effects were dose-related. Degeneration with regeneration of the olfactory epithelium (very slight to moderate) occurred in all P1 and P2 males and females exposed to 75 ppm. The degeneration consisted of thinning and disarray of the olfactory epithelial cells, which was most prevalent in the anterior and dorsal aspects of the nasal passages. Regenerative hyperplasia of the olfactory epithelium accompanied the degenerative change in multifocal sites. A lesser degree of multifocal olfactory epithelial degeneration (very slight), without accompanying regenerative hyperplasia, was noted in 7/27 P1 females exposed to 25 ppm, and in 6/27 P2 males and 8/27 P2 females exposed to 25 ppm. One P1 female and one P2 female exposed to 5 ppm also had very slight multifocal olfactory epithelial degeneration. However, the degeneration was located in only two sites of the nose for both of these animals, and therefore was interpreted to be comparable with spontaneous focal olfactory epithelial degeneration that was noted in 2/27 control group P1 females and 3/27 control group P2 females, and not an effect of treatment.

There were several histopathologic effects that accompanied the degeneration of the olfactory epithelium. Very slight or slight degeneration of the olfactory nerve was present in most of the P1 and P2 males and females exposed to 75 ppm, and one P1 male exposed to 25 ppm. This effect was characterized by thinning of the axons and reduction in the diameter of the olfactory nerve fascicles in areas of olfactory epithelial degeneration. Very slight or slight multifocal chronic-active inflammation accompanied the olfactory epithelial degeneration in 16/27 P1 males, 20/27 P1 females, 14/27 P2 males, and 8/27 P2 females exposed to 75 ppm, and in one or two males and females from both generations exposed to 25 ppm. The inflammation
consisted of neutrophils in the olfactory epithelium, with or without the presence of a mucopurulent exudate. Very slight multifocal necrosis of individual olfactory epithelial cells, with or without exfoliation of necrotic cells into the lumen of the nasal passages, was present in most of the P1 and P2 males and females exposed to 75 ppm, and a few P1 and P2 animals (one to four per sex) exposed to 25 ppm. One P1 female exposed to 5 ppm also had very slight multifocal necrosis of individual olfactory epithelial cells. However, the necrosis was located in only two sites of the nose in this animal, and therefore was interpreted to be comparable with spontaneous focal olfactory epithelial cell necrosis that was noted in one control group P1 male, one control group P1 female, and one control group P2 female, and not an effect of treatment.

A treatment-related increase in the incidence of very slight or slight multifocal hyperplasia of the transitional epithelium that covers the nasal turbinates was present in P1 and P2 males and females exposed to 25 or 75 ppm. The incidence and severity of transitional epithelial hyperplasia in P1 and P2 males and females exposed to 5 ppm was comparable to controls.

A treatment-related increase in the incidence of very slight or slight diffuse hyperplasia and hypertrophy of the respiratory epithelium that covers the nasal septum and dorsal portion of the anterior nasal cavity was present in P1 males and females exposed to 25 or 75 ppm, and in P2 males and females exposed to 75 ppm. The incidence and severity of respiratory epithelial hyperplasia and hypertrophy in P1 and P2 males and females exposed to 5 ppm was comparable to controls.

Treatment-related very slight focal or multifocal mineralization of the olfactory epithelium was present in one or two P1 and P2 animals exposed to 25 ppm, 6/27 P1 males, 4/27 P1 females, 16/27 P2 males and 14/27 P2 females exposed to 75 ppm. The mineralization was present in areas olfactory epithelial degeneration. One P1 female exposed to 75 ppm also had slight multifocal mineralization of the nasal respiratory epithelium that was interpreted to be treatment related. Other treatmentrelated nasal effects consisted of very slight multifocal squamous metaplasia of the transitional epithelium in 5/27 P1 males exposed to 75 ppm, and ulceration of the olfactory epithelium in four P1 males, one P1 female, and one P2 female exposed to
75 ppm.

All other histopathologic observations were considered to be spontaneous alterations, or caused by accidental trauma, unassociated with inhalation exposure of methyl acrylate. There were no histopathologic systemic effects in P1 or P2 rats at any exposure level. The NOEC for histopathologic nasal effects was 5 ppm.

Histopathologic Nasal Tissue Effects
P2 Males
Dose (ppm) 0 5 25 75
NASAL TISSUE - PHARYNX (number examined) (27) (27) (27) (27)
Degeneration, olfactory epithelium, focal -very slight 0 0 1 0
Degeneration, olfactory epithelium, multifocal, -very slight 0 0 6a 0
Degeneration, olfactory nerve, multifocal -very slight 0 0 0 14a
-slight 0 0 0 13a
Degeneration with Regeneration, olfactory epithelium, multifocal-slight 0 0 0 13a
-moderate 0 0 0 14a
Hyperplasia, transitional epithelium; multifocal -very slight 4 4 18a 8a
-slight 0 0 2a 19a
Hyperplasia and Hypertrophy, goblet cell, respiratory epithelium, diffuse-very slight 0 0 0 6a
-slight 0 0 0 3a
Inflammation, chronic active, olfactory epithelium, multifocal-very slight 0 0 0 14a
Mineralization, olfactory epithelium, focal -very slight 0 0 1a 1a
Mineralization, olfactory epithelium, multifocal -very slight 0 0 1a 15a
Necrosis, individual cell, olfactory epithelium, multifocal -very slight 0 0 1a 24a
Ulcer, olfactory epithelium, focal -very slight 1 0 0 1a
a-Indicates the effects judged to be treatment-related.
Histopathologic Nasal Tissue Effects
P2 Females
Dose (ppm) 0 5 25 75
NASAL TISSUE - PHARYNX (number examined) (27) (27) (27) (27)
Degeneration, olfactory epithelium, focal -very slight 3 2 4 0
Degeneration, olfactory epithelium, multifocal -very slight 0 1 8a 0
Degeneration, olfactory nerve, multifocal -very slight 0 0 0 14a
-slight 0 0 0 12a
Degeneration with Regeneration, olfactory epithelium, multifocal-very slight 0 0 0 1a
-slight 0 0 0 14a
-moderate 0 0 0 12a
Hyperplasia, transitional epithelium; multifocal -very slight 9 6 23a 25a
-slight 1 0 0 1a
Hyperplasia and Hypertrophy, goblet cell, respiratory epithelium, diffuse-very slight 7 8 11 16a
-slight 3 1 5 2
Inflammation, chronic active, olfactory epithelium, multifocal-very slight 0 0 1a 8a
Mineralization, olfactory epithelium, multifocal -very slight 0 0 1a 14a
Necrosis, individual cell, olfactory epithelium, focal -very slight 1 0 1 0
Necrosis, individual cell, olfactory epithelium, multifocal -very slight 0 0 4a 27a
a-Indicates the effects judged to be treatment-related.

Histologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effects of treatment. There were no treatment-related or statistically-identified differences in the mean number of small and growing ovarian follicles in females exposed to 75 ppm as compared to females from the control group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no evidence of an effect on estrous cyclicity at any dose level of methyl acrylate in either generation.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no treatment-related effects of methyl acrylate on any sperm analysis parameter at any exposure level in either generation.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects of treatment at any exposure level on mating, conception, fertility or gestation indices, post-implantation loss, time to mating or gestation length in either generation.

Dose descriptor:

NOEC

Remarks:

reproductive toxicity

Effect level:

ca. 0.269 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 75 ppm; the highest concentration tested.

Dose descriptor:

NOEC

Remarks:

parental local toxicity

Effect level:

0.019 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 5 ppm; based on histologic changes in nasal tissues.

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on behavior or demeanor were observed in any phase of the study at any dose level. A number of incidental observations bearing no relation to treatment were observed.

Observations made on F1 and F2 pups during their respective lactation periods revealed no effects related to treatment. Incidental findings, which included a small number of observations in the control, low-, middle-, and high-dose groups, were seen with no evidence of a dose-response relationship. Included among these incidental findings was a single high dose pup which exhibited a head tilt and circling behavior associated with overgrown incisors. This pup was euthanized on lactation day 25.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no effects of treatment at any exposure pup survival.
There were no effects of treatment on the number of pups born live, number of pups born dead, or on litter size at any time interval in any exposure group for either generation.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1 pup body weights from all exposure groups were comparable to control until PND 14. Male and female pups from the 75 ppm exposure group had decreased body weights that were statistically identified when compared to control on PNDs 14, 21, and 28. There were no treatment-related findings for F1 pup body weights from the 25 or 5 ppm exposure groups when compared to control. On PND 7, there was a statistically identified decrease in pup body weights of F1 females from the 5 ppm exposure group. This was considered spurious and unrelated to treatment due to the lack of a dose response relationship and because it was not repeated in the next generation.

Selected F1 Pup Body Weights (g)
0 ppm 5 ppm 25 ppm 75 ppm
PND 14 Males 27.0 26.7 26.3 24.3*
Percent from Control NA -1% -3% -10%
PND 14 Females 26.5 25.8 25.6 23.7*
Percent from Control NA -3% -3% -11%
PND 21 Males 44.2 42.3 42.6 39.5*
Percent from Control NA -4% -4% -11%
PND 21 Females 43.8 41.1 41.4 39.3*
Percent from Control NA -6% -6% -10%
PND 28 Males 83.1 82.2 82.0 77.3*
Percent from Control NA -1% -1% -7%
PND 28 Females 79.4 76.6 76.6 73.1*
Percent from Control NA -4% -4% -8%
*Statistically different from control mean by Dunnett’s Test, alpha = 0.05.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no effects of treatment at any exposure level on pup sex ratio in either generation.
Age at vaginal opening and age at preputial separation were similar in all exposure groups, indicating no effect of treatment on these end points despite the lower body weight of the 75 ppm animals.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The final body weights of F1 weanling males and females from the 75 ppm group were approximately 6% lower than controls, and although not statistically identified, were considered treatment-related due to the immediately preceding decrease in pup body weights from PND 14-28. There were no treatment-related alterations in organ weights of F1 weanlings at any dose level.

Final Body Weight Data – F1 Weanlings
Concentration (ppm)
0 5 25 75
Parameter MALES
Final Body Weight (g) 88.1 86.7 87.2 82.6a
Parameter FEMALES
Final Body Weight (g) 81.9 77.8 80.6 76.7a
a- Values interpreted to be treatment-related effects.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathologic observations in F1 weanlings at any exposure level.

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOEC

Generation:

F1

Effect level:

ca. 0.092 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 25 ppm; based on decreased body weights of pups at 75 ppm which were secondary to parental toxicity.

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on behavior or demeanor were observed in any phase of the study at any dose level. A number of incidental observations bearing no relation to treatment were observed.

Observations made on F1 and F2 pups during their respective lactation periods revealed no effects related to treatment. Incidental findings, which included a small number of observations in the control, low-, middle-, and high-dose groups, were seen with no evidence of a dose-response relationship. Included among these incidental findings was a single high dose pup which exhibited a head tilt and circling behavior associated with overgrown incisors. This pup was euthanized on lactation day 25.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no effects of treatment at any exposure pup survival.
There were no effects of treatment on the number of pups born live, number of pups born dead, or on litter size at any time interval in any exposure group for either generation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects on the body weights of F2 pups were similar to what was seen in the previous generation. F2 pup body weights from all exposure groups were comparable to control until PND 14. Male and female pups from the 75 ppm exposure group had decreased body weights that were statistically identified when compared to control on PNDs 14, 21, and 28. There were no treatment-related or statistical findings for F1 pup body weights from the 25 or 5 ppm exposure groups when compared to control.

These findings are likely secondary to maternal toxicity in the form of decreased maternal body weights of ~10% and severe nasal irritation. This conclusion is supported by a feed restriction study where a 10-20% decrease in maternal body weight can lead to decreased pup weights by as much as 21% (Carney, et al., 2004).

Selected F2 Pup Body Weights (g)
0 ppm 5 ppm 25 ppm 75 ppm
PND 14 Males 30.3 29.8 29.6 27.4*
Percent from Control NA -2% -2% -10%
PND 14 Females 29.7 28.7 29.0 26.7*
Percent from Control NA -3% -2% -10%
PND 21 Males 49.0 48.7 47.5 44.1*
Percent from Control NA -1% -3% -10%
PND 21 Females 48.3 46.5 46.8 42.9*
Percent from Control NA -4% -3% -11%
PND 28 Males 89.7 89.7 87.5 83.4*
Percent from Control NA 0% -2% -7%
PND 28 Females 84.2 82.3 82.0 77.8*
Percent from Control NA -2% -3% -8%
*Statistically different from control mean by Dunnett’s Test, alpha = 0.05.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no effects of treatment at any exposure level on pup sex ratio in either generation.
Age at vaginal opening and age at preputial separation were similar in all exposure groups, indicating no effect of treatment on these end points despite the lower body weight of the 75 ppm animals.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

F2 weanling males and females from the 75 ppm group had treatment-related lower final body weights (statistically identified in females at 75 ppm). The final body weights of F2 weanling males and females from the 75 ppm group were 5.8% and 7.7% lower than controls, respectively. As in the 75 ppm group F1 pups, these decreases in final body weights (PND 29) were reflective of the statistically significant decreases in pup body weights during the preceding two weeks. There were no treatment-related alterations in organ weights of F2 weanlings at any dose level. The only statistically identified organ weight alteration was a higher absolute brain weight in F2 male weanlings from the 5 ppm exposure group, which was unrelated to treatment due to the lack of a dose response.

Final Body Weight Data – F2 Weanlings
Concentration (ppm)
0 5 25 75
Parameter MALES
Final Body Weight (g) 94.6 93.0 94.6 89.1a
Parameter FEMALES
Final Body Weight (g) 87.2 84.7 84.5 80.4*a
*Statistically different from control mean by Dunnett’s Test, alpha = 0.05.
a- Values interpreted to be treatment-related effects.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In F2 weanlings, 2/81 males and 3/78 females from the 75 ppm exposure group had necrosis of the tail. This observation may have been related to treatment, but the significance of the tail necrosis is not known. All other gross pathologic observations from F1 and F2 weanlings were considered to be spontaneous alterations, unassociated with exposure to methyl acrylate.

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOEC

Remarks:

developmental toxicity

Generation:

F2

Effect level:

ca. 0.092 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 25 ppm; based on decreases in pup at 75 ppm which were secondary to parental toxicity.

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

0.269 mg/L air (analytical)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Screening Test Results

Treatment-related clinical signs in the 150 ppm P1 males and females were limited to a transient sneezing/huffing sound noted each day immediately following the end of exposure, occurring from day 36. This sound was observed until termination of the P1 males. In females, this sound was no longer present when exposure was stopped from GD 21 – LD 4, but was observed again upon resumption of exposure (LD 5-28),

albeit at a lesser severity and incidence.

 

P1 males and females exposed to 150 ppm had treatment-related decreases in body weights, body weight gains and feed consumption that were observed during the pre-breeding, gestation and lactation phases. Similar, but less severe effects on body weight and feed consumption were seen in males and females exposed to 75 ppm. There was a dose-dependent decrease in the terminal body weights of rats exposed to methyl acrylate.

 

There were no treatment-related effects on any reproductive parameters, organ weights, or gross pathology.

 

Dose-related histopathologic effects were present in the nasal tissues of males and females at all exposure concentrations. Degeneration with regeneration of the olfactory epithelium (very slight to severe) occurred in males and females at 150 ppm and in males at 75 ppm. Regenerative hyperplasia of the olfactory epithelium accompanied the degenerative change in multifocal sites. A lesser degree of olfactory epithelial degeneration (very slight), without accompanying regenerative hyperplasia, was noted in females at 75 ppm, and in males and females at 25 ppm. Very slight or slight degeneration of the olfactory nerve was present in males and females at 150 ppm only. Very slight or slight chronic active inflammation accompanied the olfactory epithelial degeneration in males and females at 150 ppm, and in females at 75 ppm. Very slight necrosis of individual olfactory epithelial cells, and multifocal, very slight or slight hyperlasia of the transitional epithelium that covers the nasal turbinates was present in rats exposed to 25, 75, or 150 ppm.

 

There was a slight decrease in the PND 14 body weight of pups whose dams were exposed to 150 ppm methyl acrylate.

 No clinical signs were observed in the F1 males or females that were exposed from PND 28-35. F1 males and females exposed to 150 ppm had treatment-related decreases in body weights and feed consumption. Similar, but less severe effects on body weight and feed consumption were seen in F1 males and females exposed to 75 ppm.

 

Percent Difference in Terminal Body Weight Compared to Control

25 ppm 75 ppm 150 ppm

P1 Males -1% -7% -13%

P1 Females -1% -2% -12%

F1 Males +2% -4% -18%

F1 Females -1% -9% -17%

Chamber Concentration

Mean chamber concentration values during the study were 0, 5.3 ± 0.2, 25.7 ± 0.3, and 75.4 ± 0.6 ppm. Actual mean chamber concentration values deviated 0.5-6% from the targeted values of 0, 5, 25, and 75 ppm.

 

In-Life Observations

Examinations performed on all animals prior to the study start revealed that all animals were in good health for study purposes.

Conclusions:

The no-observed-effect concentration (NOEC) for parental systemic toxicity was determined to be 5 ppm and was based on histologic changes in the nasal tissues seen at higher concentrations. The NOEC for developmental toxicity was 25 ppm, based on decreases in pup body weight at 75 ppm which were secondary to parental toxicity. The NOEC for reproductive toxicity was 75 ppm, the highest concentration tested.

Executive summary:

The purpose of this two-generation inhalation reproduction toxicity study was to evaluate the potential effects of methyl acrylate on male and female reproductive function, as well as the survival, growth and development of the offspring. Groups of 27 male and 27 female Crl:CD(SD) rats were whole-body exposed to target concentrations of 0, 5, 25, or 75 ppm vaporized methyl acrylate for six hours/day, seven days/week, resulting in actual average concentrations of 0, 5.3 ± 0.2, 25.7 ± 0.3, and 75.4 ± 0.6 ppm, respectively. Rats were exposed daily for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation for two generations. Maternal rats were not exposed after GD 20 through LD 4 in order to allow for parturition and initiation of lactation. Exposure of maternal rats continued from LD 5 – LD 28. In-life parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In addition, post-mortem evaluations included gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.

Treatment-related effects in parental rats exposed to 75 ppm included decreased body weight and feed consumption in males and females throughout most of the two generation study. There were no effects on body weight or feed consumption at 25 or 5 ppm.

Treatment-related, adverse histopathologic effects were present in the nasal tissues of P1 and P2 males and females exposed to 25 or 75 ppm. The incidence and severity of the nasal effects were concentration dependent. Degeneration with regeneration of the olfactory epithelium (very slight to moderate) occurred in all P1 and P2 males and females exposed to 75 ppm. Very slight olfactory epithelial degeneration, without accompanying regenerative hyperplasia, was noted in some of the P1 and P2 females and P2 males exposed to 25 ppm. There were several histopathologic effects that accompanied the degeneration of the olfactory epithelium. Very slight or slight degeneration of the olfactory nerve was present in most of the P1 and P2 males and females exposed to 75 ppm, and one P1 male exposed to 25 ppm. Very slight or slight chronic-active inflammation was present in 16/27 P1 males, 20/27 P1 females, 14/27 P2 males, and 8/27 P2 females exposed to 75 ppm, and in one or

two males and females from both generations exposed to 25 ppm. Very slight necrosis of individual olfactory epithelial cells was present in most of the P1 and P2 males and females exposed to 75 ppm, and a few P1 and P2 animals (one to four per sex) exposed to 25 ppm. Very slight mineralization of the olfactory epithelium was present in one or two P1 and P2 animals exposed to 25 ppm, and in 6/27 P1 males, 4/27 P1 females, 16/27 P2 males and 14/27 P2 females exposed to 75 ppm. Other nasal effects consisted of an increase in the incidence of very slight or slight hyperplasia of the transitional epithelium in P1 and P2 males and females exposed to 25 or 75 ppm, an increase in the incidence of very slight or slight hyperplasia and hypertrophy of the respiratory epithelium in P1 males and females exposed to 25 or 75 ppm, and in P2 males and females exposed to 75 ppm, very slight squamous metaplasia of the transitional epithelium in 5/27 P1 males exposed to 75 ppm, and

ulceration of the olfactory epithelium in four P1 males, one P1 female, and one P2 female exposed to 75 ppm. There were no treatment-related histopathologic effects in P1 or P2 animals exposed to 5 ppm.

No treatment-related effects were seen in reproductive function or pup survival. However, pup body weights of the 75 ppm exposure group were decreased on PND 14-28 in both generations. There were no effects on pup body weight in rats exposed to 25 or 5 ppm. The effects on pup body weight, as well as the changes in parental body weight and feed consumption, likely were secondary changes all stemming from nasal irritation and resultant stress.

In summary, the no-observed-effect concentration (NOEC) for parental systemic toxicity was determined to be 5 ppm and was based on histologic changes in the nasal tissues seen at higher concentrations. The NOEC for developmental toxicity was 25 ppm, based on decreases in pup body weight at 75 ppm which were secondary to parental toxicity. The NOEC for reproductive toxicity was 75 ppm, the highest concentration tested.

Reference 7

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 1992 to 19 Apr 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: ≥98.9%

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar rats (Chbb = THOM (SPF))
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: (P): 35 ± 1 days
- Weight at study initiation: (P) Males: 140.0 (127 - 154) g; Females: 118.8 (106 - 130) g
- Housing: Single in type DK III stainless steel wire mesh cages, with the following exceptions: during mating periods, the males designated for mating were kept individually in Makrolon cages, type M III; for the overnight mating the females were put into the cages of the males. From day 18 of pregnancy until day 14 after birth, the pregnant animals and their litters were also housed in Makrolon type M III cages.
- Diet (ad libitum): Kliba maintenance diet rat/ mouse/hamster GLP 343 meal (KLINGENTALMUEHLE AG, Kaiseraugst, Switzerland)
- Water (ad libitum): Tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 3 weeks.
- Proof of pregnancy: sperm in vaginal smear referred as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content of Acrylic acid in the aqueous solutions was determined by gas chromatography.

Duration of treatment / exposure:

Exposure period: F0: at least 70 days before the mating and afterwards during the gestation and lactation periods.
F1: at least 98 days before the mating and afterwards during the gestation and lactation periods.
Premating exposure period (males): at least 70 days
Premating exposure period (females): at least 70 days
Duration of test: approx. 12 months

Frequency of treatment:

continuously

Dose / conc.:

500 ppm (nominal)

Remarks:

corresponding to approx. 53 mg/kg bw/day

Dose / conc.:

2 500 ppm (nominal)

Remarks:

corresponding to approx. 240 mg/kg bw/day

Dose / conc.:

5 000 ppm (nominal)

Remarks:

corresponding to approx. 460 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Positive control:

none

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
All parental animals were checked daily for clinically evident signs of toxicity. Particular attention was given to the nesting, littering and lactation behaviour of the dams, but only special findings were documented.

BODY WEIGHT:
- Parental animals: generally, body weight was determined once weekly until the end of the study, and at the time of necropsy.
- F0 and F1 fertilized females and females with litter: body weight was determined on the day of sperm evidence in the vaginal smear and thereafter on days 7, 14 and 20 of gestation, one day after parturition, and on days 7, 14 and 21 post-parturition.
- Females without positive evidence of sperms: body weight was not determined during the mating interval.
- Females without litter: body weight was not determined during the lactation phase.

FOOD CONSUMPTION:
- F0 and F1 parental animals: food consumption was determined once weekly (over 7 days) during the period prior mating.
- Pregnant females: food consumption was determined for days 0-7, 7-14, 14-20 post coitum (pc).
- Lactating females: food consumption was determined for days 0-4, 4-7, 7-14 post parturition (pp).
- F0 and F1 dams between day 14 and 21 pp: food consumption was not determined for the F0 and F1 dams between day 14 and 21 pp, since during this period the pups started consumption of solid food; therefore there was no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: food consumption was not determined respectively during mating period, gestation period or lactation phase.

WATER CONSUMPTION:
- F0 and F1 parental animals: water consumption was determined once weekly (over 3 days) during the period prior mating.
- Pregnant females: water consumption was determined for days 0-1, 6-7, 13-14, 19-20 post coitum (pc).
- Lactating females: water consumption was determined for days 1-2, 3-4, 6-7, 13-14 post parturition (pp).
- F0 and F1 dams from day 20 and 21 pp: water consumption was not determined for the F0 and F1 dams for days 20 - 21, since during this period the pups started consumption of water; therefore there was no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: water consumption was not determined respectively during mating period, gestation period or lactation phase.

INTAKE OF TEST SUBSTANCE:
The intake of test substance (IT, in mg/kg bw/day) was calculated according to the following formula:

ITx = WCx * D / BWy
D = dose in ppm
WCx = daily water consumption on day x; in g
BWy = body weight on day y; in g

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

The pups (F1 and F2 litters) were examined as soon as on their day of birth for the determination of the total number of pups and the number of liveborn and stillborn pups (pups died on day of birth prior the first examination). Thereafter the pups were checked twice daily on workdays (once a day on week ends and public holidays) for mortality (i.e. dead and moribund pups) and the mortality (number and percentage) was determined for the day of birth (i.e. day 0) and for the periods: days 1 - 4, 5 - 7, 8 - 14 and 15 - 21 of lactation. Pups that died accidentally and had to be sacrificed because of maternal death were not considered for calculation. The number of surviving pups was determined for days 0, 4, 7, 14 and 21 of lactation and served for the calculation of the viability index and the lactation index.

The sex of the pups was determined on day 0 and day 21 (measurement of the anogenital distance, which is known to be greater in male pups than in females), and the sex ratio was calculated according to following formula:

- Sex ratio = number of live male or female pups on day 0/21 * 100 / number of live male and female pups on day 0/21

The pups were weighed on days 1, 4, 7, 14 and 21 after birth, and they were examined daily for clinical symptoms or gross morphological abnormalities. The determination of the relative organ weight was based on the pup body weight on day 21 after birth. The bodies of the sacrificed pups were examined for external abnormalities and the organs also were subjected to gross pathology; skeletal staining according to the modified Dawson´s method and/or further processing of the head according to Wilson´s method was done in case of abnormal findings. Stillborn pups as well as pups that died during weaning also were subjected to necropsy.

Development stages / Behavioral tests:
Physical development was assessed by monitoring pinna unfolding, opening of the auditory canal and opening of the eyes. Additional tests
were performed to assess grip reflex, hearing and pupillary reflex as follows :
- Grip reflex: Tested on day 13 after birth by placing front paws onto 3-mm diameter rod. For a positive response, the animal had to grip the bar and pull itself up.
- Hearing test: On day 21 after birth, animals were placed in a soundproof box and exposed to a sound (0 .1 sec, 5000 Hz, about 90 dB); a startle reflex was considered a response to this stimulus.
- Pupillary reflex: On day 2l after birth, pupillary constriction reflex was assessed by shining a penlight on the eye and observing the reaction.

Postmortem examinations (parental animals):

SACRIFICE
Parental animals were killed by decapitation under CO2 anaesthesia and examined macroscopically.

GROSS NECROPSY
Terminal body weights as well as the weights of liver, kidneys, epididymides and testes were recorded .

HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, kidneys and stomach (non-glandular and glandular).

Postmortem examinations (offspring):

SACRIFICE
Pups were killed by CO2 asphyxiation, examined externally, eviscerated and their organs assessed macroscopically.

GROSS NECROPSY
External and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
Vagina, cervix, uterus, ovaries, oviducts, testes, epididymides, seminal vesicles, coagulation gland, oesophagus and duodenum.

Statistics:

The statistical assessment of the different data obtained within the present study was based on following methods, depending on the parameters considered: Dunnett test, Fisher`s exact test and Wilcoxon test.

Reproductive indices:

Mating and fertility indices were calculated according to following formulas:

- Male mating index (%) = number of males with confirmed mating * 100 / number of males placed with females

- Male fertility index (%) = number of males proving their fertility * 100 / number of males placed with females

Remark:
Males were defined as “with confirmed mating” by the presence of vaginal sperm in the female, or by the production of a litter, or by the presence of fetuses in the uterus.
Males were defined as “proving their fertility” by female giving birth to a litter or having pups or fetuses in the uterus.

Re-evaluation of fertility:
If an animal of the F0 or F1 generation parental animals had not produced any offspring after the scheduled mating of F0 parents (to get F1 litter) or after the scheduled mating of F1 parents (to get of F2 litter), those animals treated with the test substance were mated with fertile animals of the control groups. Animals of the control groups which seemed to be infertile were mated with mating partners with proven fertility of the controls.
After fertility had been reevaluated, the animals were sacrificed and subjected to gross-pathological and histopathological assessments. The uteri of the females reevaluated for fertility were examined for live and dead implantations. In the case of an apparently non-pregnant animal or of an empty uterus horn in the case of single-horn pregnancy, the uterus was stained with sodium sulfide and assessed for early implantations. Then the uteri were rinsed carefully under running water. After these examinations were completed, the uteri were transferred to the pathology lab for further fixation and evaluation.

Offspring viability indices:

- Viability index (%) = number of live pups on day 4 after birth * 100 / number of liveborn pups on the day of birth
- Lactation index (%) = number live pups on day 21 after birth * 100 / number of live pups on day 4 after birth

Remark:
Day 4 after birth preceded standardization of the litters.
Day 21 after birth followed standardization of the litters.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs which might be attributed to the test substance were detected in male or female P0 generation parental animals. The 3 concentrations administered in the drinking water did not lead to disturbances of the general behavior in any of the P0 parental animals.
There were no particular substance-related clinical findings in P0 females during the gestation period for P1 litter. Insufficient nesting activity was observed for several dams of all groups including the controls.
No substance-related clinical findings were recorded for the P0 dams during the P1 lactation period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortalities in any of the P0 generation parental animals in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the P0 males statistically significant reductions in mean body weights were seen in the highest dose group (5000 ppm) from week 12 until week 20 of the study period. Body weight changes of the high dose males were statistically significantly diminished only at certain study intervals (weeks 0-1, 6-7, 11-12); if calculated for the total study period (weeks 0-20), body weight gain of the high dose males was about 9% lower than that of the respective controls.
Body weights and weight gains of the substance-treated females were similar to control values during the premating period and during gestation and lactation. Only during the first week of gestation did the high dose dams gain statistically significantly less weight than the corresponding controls.
Finally the impairments in body weight/body weight gain in the high dose P0 males and - to a lesser extent - in the P0 females are assessed as being substance-related effects. All other statistically significant differences in body weights and body weight gains are considered unrelated to the test substance because the values were not influenced in a dose-dependent manner and/or are within the biological range of variation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In general, the food consumption of the males (during the premating period) and of the females (during premating, gestation and lactation periods) of all test groups was not influenced by the test substance administration. It was, however, slightly, but statistically significantly reduced in the high dose males during the first week of the premating period and in the females of 5000 ppm test group during the second week of the lactation period.
The sporadic and only marginal reductions in food consumption of the 5000 ppm rats are probably related to the reduced consumption of aqueous acrylic acid solutions of these animals and thus are likely indirectly associated with the administration of the test substance. All other observable differences between the groups are without biological relevance, because they are not dose-related; this includes the statistically significantly increased food consumption of the low dose females during study weeks 0-1 and 6-7 of the premating period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

The water consumption of the high dose male and female animals was clearly reduced. This reduction was statistically significant during the premating period. It was also diminished in the 5000 ppm females during gestation and lactation of P1 litter. In total the 5000 ppm males consumed about 11% and the high dose females about 13% less drinking water (aqueous acrylic acid solutions) then the respective controls during the first 10 study weeks. The marked reduction in the drinking water consumption of the high dose rats was associated with the test substance administration. All other observable differences between the groups in respect to water consumption are without biological relevance, because they are not dose-related; this includes the statistically significantly increased water consumption of the 500 ppm female animals during premating weeks 6-7 and 9-10.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Males: For all P0 males which were placed with females to generate F1 pups mating was confirmed; thus, the male mating index was 100% in all groups. For nearly all P0 males fertility could be confirmed within the scheduled mating interval; the fertility index varied between 92% and 96% with no treatment related effect. Thus, the fertility of the P0 generation parental males was not adversely influenced by the administration of aqueous acrylic acid solutions.

Females: The female mating index calculated after the mating period for F1 litter was 100% for all groups. The mean duration until sperm was detected (day 0 pc) varied between 1.8 and 3.8 days and was statistically significantly longer for the high dose dams; the high dose value (3.8 days), however, is substantially similar to the mean cohabitation time value of the control group (3.2 days) of the second parental generation (P1 animals) and therefore was not considered treatment-related. Only one or two females in all groups, including the controls, did not become pregnant within the scheduled mating interval. The fertility index varied between 92% and 96% without any dose-response relationship. All females in question except the 2 low dose females proved to be fertile after being mated again with control males. The mean duration of gestation was similar in all groups and the gestation index reached 100% for all groups. The mean number of pups delivered/dam was uninfluenced by the test substance administered. The number of liveborn and stillborn pups was comparable between the groups, and the live birth index was 98% in test groups. Thus, the administration of aqueous acrylic acid solutions did not adversely affect reproduction and delivery data of the P0 generation parental females.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

240 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

460 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs which might have been attributed to the test substance administered were detected in male or female P1 generation parental animals. The 3 doses administered in drinking water did not lead to disturbances of the general behavior in any of the P1 parental animals. One male animal of 2500 ppm test group developed a severe skin lesion on the base of the tail and was sacrificed in a moribund state. Another male of the same dose group showed unilateral chromodacryorrhea. The clinical findings which occurred in just two intermediate dose males were spontaneous in nature.

No particular clinical findings were noted for P1 dams with positive sperm detection except insufficient or no nesting activity, which was recorded for several dams of all groups (0, 500, 2500 and 5000 ppm) and which occurred without a clear dose-response relationship. One female of the low dose group showed vaginal hemorrhage towards or after the end of the gestation period (days 23 - 26 pc), and was not able to deliver the pups, which were palpable earlier in the abdomen of this dam. After day 26 pc, no pups could be palpated for this dam.

There were no substance-related clinical findings in the P1 dams during the lactation of F2 litters. Only one dam of the low dose group and one dam of the high dose group did not nurse their pups properly; all pups of high dose dam were cannibalized and/or died intercurrently. Furthermore, another low dose dam showed blood in bedding during the first days of the lactation period, and was not able to deliver its litter completely. It delivered only 2 pups which were cannibalized on day 1 pp.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal of 2500 ppm test group had to be sacrificed in a moribund state due to a severe skin lesion on the base of the tail. There were no other unscheduled mortalities in any of the test groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the P1 males, statistically significant reductions in mean body weights were seen in the highest dose group (5000 ppm) throughout the total study period (about 87% of the control value at the end of this study interval). Body weight gains of the 5000 ppm males, however, were generally similar to the respective control values. In total, the weight gain of the high dose P1 males was only about 5% lower than the body weight gain of the control males. Body weights of the 5000 ppm females were also statistically significantly reduced during the premating period (about 89% of the control value at the end of the premating phase). During gestation and lactation of F2 litter, mean body weights of the high dose P1 dams were statistically significantly lower than the corresponding control values. During premating, gestation and lactation periods body weight gain of the high dose females reached or even exceeded body weight gain of the controls.
The statistically significantly lower body weights recorded for the 5000 ppm P1 males and females were considered to be related to the test substance administration. A lower body weight was also recorded for these animals at the pup stage; during the following premating period the P1 parental animals of the high dose group gained substantially as much weight as the controls, but the body weights of the 5000 ppm rats were still reduced. All differences between the controls and 500 or 2500 ppm groups concerning body weights/weight gains, however, were regarded as spontaneous in nature.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of the males and females of 5000 ppm test group was clearly reduced during the premating period, the differences in comparison to the controls being statistically significant at most time intervals. In total, the high dose males consumed about 9% and the females about 8% less food than the respective control animals during the premating phase. Food intake was also statistically significantly diminished in the females of this test group (5000 ppm) during the gestation period (days 7-20 pc) and during the lactation period (days 7-14 pp only). The reduction in food consumption of the 5000 ppm males and females was considered to be related to the administration of the test substance. All other differences in food consumption between the groups are without any biological relevance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

In comparison to the respective control values the water consumption of the 2500 and 5000 ppm F1 males and females was distinctly lower during the premating period, the differences being statistically significant at all intervals in the high dose level and in several but not all intervals at the intermediate dose. In total a clear dose-response relationship was observed: high dose males consumed about 18%, intermediate dose males about 9% less water than control males; for high dose females about 27% and for intermediate dose females about 13% less water intake than in the female controls was recorded. Water consumption was also reduced during gestation and lactation periods in these test groups, again more pronounced in the high than in the 2500 ppm group. The water consumption of the animals of the low dose group (500 ppm) reached or even exceeded the relevant control values during premating, gestation and the lactation periods. The distinct reductions in the drinking water consumption in both sexes at 2500 and 5000 ppm are considered treatment-related, whereas the differences in water consumption between the low dose group and the control are considered to be without toxicological relevance.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Males: For all P1 males which were placed with females to generate F2 pups, mating was confirmed. The male mating index was 100% in all groups.

Females: The female mating index reached 100% in all groups. The mean duration until sperm was detected (day 0 pc) varied between 2.1 and 3.2 days and was highest for the control group, because one dam of this group had a prolonged cohabitation time. In the scheduled mating interval (F2), 2 control females did not become pregnant. Therefore, the fertility index was lowest in the control group (92%), whereas it was 100% in all substance-treated groups. There were no biologically relevant differences between test groups and the controls concerning the mean duration of gestation and the number of liveborn and stillborn F2 pups. All pregnant females - except one low dose female which had palpable pups in the uterus but did not deliver - gave birth to litters with liveborn pups. Consequently, the gestation and the live birth indices were not influenced by the administration of the test substance. The mean number of delivered pups/dam was not influenced by the test substance administered.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

53 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

440 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

None of the F1 pups of any one group showed abnormal clinical findings during the lactation period.
There were no biologically relevant differences between the control and the substance-treated F1 pups in the several morphological development stages monitored up to weaning.
No remarkable differences between the groups were observed in the different behavioral tests which the pups underwent up to weaning.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No substance-induced effects on pup mortality/viability were recorded during the lactation period. Both the viability index, as an indicator of the viability of the pups during the first 4 days after birth, and the lactation index, as an indicator how the pups were nursed during the rest of their rearing, do not show differences of biological relevance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of F1 male and female pups were clearly reduced in 5000 ppm test group from day 14 pp onwards and impaired in the intermediate dose (2500 ppm) on day 21 pp when compared to the controls. On day 21 pp the pup weights (both sexes combined) in the high dose group were about 35% and those of the 2500 ppm pups about 11% lower than the corresponding control values. Body weight gains of the 2500 ppm and 5000 ppm pups were also statistically significantly decreased from days 7 (5000 ppm) or 14 pp (2500 ppm) up to weaning (day 21 pp). The reductions in pup body weights/body weight gains in the 5000 and 2500 ppm groups were attributed to the test substance administration. All other differences concerning pup body weights/body weight gains are without any biological relevance and lie within the biological range of variation.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and on day 21 post parturition (pp) did not show any substantial difference between controls and treated groups; all differences observed are regarded as spontaneous.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Only spontaneous findings were seen at necropsy (e.g. incisors sloped, hernia diaphragmatica, dilated renal pelvis) in very few of the pups examined. All findings were present in the concurrent control at a comparable frequency and/or did not show a clear relation to dosing.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

53 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F2 generation pups did not show any clinical signs up to weaning which could be attributed to the treatment. Hydrocephaly, which occurs also occasionally in control pups was recorded in one 500 ppm pup.
Development stages: There was a statistically significantly lower incidence of F2 pups/litter with auditory canal opening on time in the intermediate dose group and with eye opening on time in the 5000 ppm group. The relevant values were within the historical control ranges and a clear relation to dosing was not observed. These effects must be considered in conjunction with the retarded weight gain of these pups and were therefore assessed as being possibly substance-related. There were no differences of biological relevance in different stages of development between the low dose and the control pups.
No substantial differences could be noted between the F2 pups of all test groups and the control pups in the different behavioral tests. The observable differences were without biological relevance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of delivered F2 pups/dam in the treatment groups was similar to the relevant control value; moreover, the percentages of liveborn and stillborn F2 pups were comparable between the groups; all differences between the groups are in the range of biological variation.
During the lactation period a statistically significant increase in pups cannibalized by dams was noted in the high and intermediate dose groups. The increased cannibalization rate was predominantly caused by just one intermediate dose dam and two high dose dams. One Female of the high group, however, neglected her pups during the lactation period, thus nearly all pups of this dam died before schedule and/or were cannibalized. Occasionally insufficient nursing behavior and cannibalism occured also in control females, and thus the higher rate of cannibalized pups at these dose levels was not considered treatment-related.
There were no differences in biological relevance between the control and the 500, 2500 and 5000 ppm F2 pups concerning viability and mortality; consequently the viability and lactation indices were not affected by the test substance administration (although some statistically significant differences existed). All relevant values are inside the historical control range and/or do not show a clear relation to dosing; moreover it had to be taken into consideration, that the high dose dams delivered on average distinctly more pups (14.0 pups/dam) than the controls (12.9 pups/dam).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights/body weight gains of the F2 male and female pups of the 5000 ppm and 2500 ppm groups were clearly influenced by the test substance administration. Mean pup body weights of the 5000 ppm pups were statistically significantly lower than the corresponding control values from days 14 (males and females) until weaning on day 21 pp, when the high dose pups (both sexes combined) weighed about 32% less than the controls. Mean pup body weights of the 2500 ppm pups were statistically significantly (about 12%) lower than the corresponding control values on day 21 pp (both sexes combined). Weight gains of the pups of the 2500 and 5000 ppm test groups were also statistically significantly reduced from the second week of the lactation period onward, the reduction more pronounced in the 5000 ppm than in the 2500 ppm pups. All differences between the control group and the 500 ppm group concerning pup body weight data of the F2 generation were considered spontaneous in nature.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

No remarkable differences between the control and the substance-treated groups were found in respect to the sex ratio of the F2 pups. The observable differences are in the range of biological variation.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examinations of F2 pups at necropsy did not reveal any differences considered to be of biological relevance between the controls and the substance-treated groups either in the type or in the number of pup necropsy observations. A few pups of the different groups showed some spontaneous findings like hernia diaphragmatica, incisors sloped, dilated renal pelvis, hydroureter, hydrocephaly, focal liver necrosis, cardiomegaly, septal defect and post mortem autolysis.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

53 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

240 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Mean Body Weight Changes (F0 parental animals), grams

Treatment week	0 mg/kg bw/d		53 mg/kg bw/d		240 mg/kg bw/d		460 mg/kg bw/d
	male	female	male	female	male	female	male	female
0 - 1	52.6	23.8	52.2	24.8	52.3	23.6	46.7^**	23.1
1 - 2	53.7	22.1	55.5	22.0	54.4	22.3	51.6	21.8
2 - 3	47.2	16.2	45.8	17.2	46.9	17.5	44.8	14.6
3 – 4	35.4	12.9	34.8	17.1^*	35.1	14.9	32.4	17.8^*
4 – 5	29.1	15.7	27.6	13.3	28.6	12.6	27.0	13.7
5 – 6	21.0	9.4	21.8	8.8	23.3	12.5	23.2	11.0
6 – 7	23.2	8.5	24.7	11.7	21.2	10.1	19.3^*	10.7
7 – 8	20.4	7.7	17.7	10.3	19.8	7.8	18.5	8.6
8 – 9	19.3	9.8	19.1	7.1	18.0	7.2	16.6	8.1
9 – 10	13.1	4.7	13.0	4.5	14.6	7.9^*	11.9	6.0
10 – 11	-7.0		-2.8		-2.8		-2.8
11 – 12	21.8		18.5		18.4		14.7^**
12 – 13	14.8		13.3		13.7		11.1
13 – 14	8.0		10.7		9.7		6.1
14 – 15	9.2		7.0		7.5		8.1
15 – 16	10.5		10.4		8.0		9.9
16 – 17	5.7		7.9		7.3		4.6
17 – 18	2.7		4.6		1.7		1.4
18 – 19	1.1		0.2		1.3		1.6
19 - 20	4.1		6.7		5.8		5.0

^*P<0.05

^**P<0.01

Reproduction and litter data for F0 parents /F1 pups

	0 ppm	500 ppm	2500 ppm	5000 ppm
Parents
Females mated	25	25	25	25
Females pregnant	24	23	23	24
Females with delivery	24	23	23	24
Mean duration of gestation (d)	22.0	21.9	21.9	21.9
Litter means
Live births/litter	13.8	13.8	13.7	14.3
Survivors day 4 preculling	13.3	13.5	13.4	13.8
Survivors day 4 postculling	7.5	7.9	7.9	7.9
Survivors day 21	7.5	7.9	7.8	7.8
Weight at day 1 (g) M/F	6.4/6.1	6.6/6.2	6.5/6.2	6.5/6.2
Weight at weaning (g) M/F	52.3/50.01	52.1/49.4	46.6^**/44.6^**	34.2^**/32.7^**
Sex ratio of live newborns % M/F	51/49	49/51	55/45	53/47
Selected as parents for the next generation M/F	25/25	25/25	25/25	25/25

^**P≤0.01

Reproduction and litter data for F1 parents /F2 pups

	0 ppm	500 ppm	2500 ppm	5000 ppm
Parents
Females mated	25	25	25	25
Females pregnant	23	23	23	24
Females with delivery	23	23	23	24
Mean duration of gestation (d)	22.0	21.9	21.9	21.9
Litter means
Live births/litter	12.5	11.6	12.0	13.8
Survivors day 4 preculling	11.8	10.6	10.8	12.5
Survivors day 4 postculling	7.7	7.0	7.5	7.8
Survivors day 21	7.7	6.9	7.5	7.4
Weight at day 1 (g) M/F	6.3/5.9	6.5/6.2	6.1/5.8	6.4/6.1
Weight at weaning (g) M/F	50.4/48.4	52.1/49.4	44.6^**/42.4^**	34.5^**/33.2^**
Sex ratio of live newborns % M/F	47/53	55/45	53/47	49/51

^**P≤0.01

Development landmarks in the F2 (mean % of pups reaching criteria/litter)

Parameter	0 ppm	500 ppm	2500 ppm	5000 ppm	Historical control range
Pinna unfolding	93.6 (16.65)	93.5 (20.87)	80.7 (33.37)	86.9 (26.87)	74-100
Auditory canal opening	98.8 (3.87)	95.1 (20.90)	91.4^*(18.09)	94.2 (12.26)	81-100
Eye opening	93.2 (18.08)	92.4 (21.89)	92.4 (21.89)	86.5^*(21.15)	85-100

^*P≤0.05

Figures in parentheses indicate standard deviations.

Reference 8

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Assessing the effect of EG on fertility and general reproductive performance in male and female rats.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: young adult nulliparous Fischer 344 rats
- Housing: two per cage in stainless-steel wire cages; during mating, each male was housed with 2 females; after mating and during lactation, the females were housed individually in plastic showbox cages with hardwood chips for nesting.
- Diet: Purina Formulab, ad libitum
- Water: city water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Photoperiod (hrs dark / hrs light): 12 /12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Administration of EG to the F0 rats of both sexes started at approximately 7 weeks of age.
Fresh diet was prepared every 2 weeks with the percentage of test item (EG) adjusted, based on the group mean body weight and food consumption, so as to maintain a relatively constant dosage level. However, the concentration of EG in the diet was not changed during gestation or during the first week of lactation, but was reduced two- and three-fold during the second and third weeks of lactation, respectively, to adjust for increased food consumption by the dams. This change in concentration was based on earlier unpublished results from the laboratory. Increased food consumption during lactation has since been reported in another study performed at the laboratory.

Details on mating procedure:

At approximately 100 days of age, 10 males were added to 20 females in each dosage group. The F1 and F2 rats were treated as described for the F0 animals until approximately 100 days of age, at which time the animals were cohabited. Brother and sister matings were avoided for each generation.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

3 generations

Frequency of treatment:

daily

Details on study schedule:

The date of parturition and the number of live and dead newborn were recorded for each litter. The appearance and behavior of dams and pups were observed daily. Litter size was randomly reduced to 10, if necessary, on Day 4 postpartum. Offspring were weighed as litters at 4 and 14 days and individually at 21 days postpartum, the day they were weaned. F1 rats were randomly selected within each dosage group for the next mating. Each litter was represented except for those conceived very late in the mating period.

Dose / conc.:

40 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

30

Control animals:

yes, concurrent no treatment

Details on study design:

Two untreated diet control groups, designated 0.0A and 0.0B, were included to estimate the variation between 2 groups treated alike.

Parental animals: Observations and examinations:

Body weights and diet consumption were recorded weekly except during gestation and lactation.

Litter observations:

Offspring were weighed as litters at 4 and 14 days and individually at 21 days post partum, the day they were weaned. F1 rats were randomly selected within each dosage group for the next mating.

Postmortem examinations (parental animals):

Necropsies were performed on five males and five females randomly selected from each dosage level of the F2 parents and the F3 weanlings. Microscopic examinations were performed on sections of liver, kidneys, lung, heart, adrenals, thyroid, trachea, accessory sex glands, adipose tissue, lymph nodes, pituitary, thymus, and testes and epididymis, or uterus and ovaries.

Statistics:

Continuous data such as body weights were compared by analysis of variance validated by Bartlett's test for homogeneity of variance. Duncan's multiple range test was used to identify individual mean differences when indicated by a significant F value. Where Bartlett's test indicated heterogeneous variances, t tests for equal or unequal variances were used to delineate differences between groups. Pup weights were compared by the method of Weil (Weil, 1970). Discontinuous data such as implantations and reproductive indices were compared by a multiple sum of ranks test. Frequency data were compared by the X2 test and by Fisher's exact test. The following reproductive indices were calculated and evaluated statistically by the previously described non parametric methods: fertility index (male and female), days from first mating to parturition, gestation index (fraction of pregnancies that resulted in litters with live pups), gestation survival index (fraction of newborn pups alive at birth), 0 to 4-day survival index, 4 to 14-day survival index, 4 to 21day survival index. The last four indices are summarized in the tables as means for ease of understanding and presentation, although the nonparametric statistical methods did not include a comparison of means.

Reproductive indices:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

Throughout the study there was no effect of EG treatment on body weight gain or diet consumption, nor was there any mortality among parental rats.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no reproductive effects associated with EG treatment doses up to 1000 mg/kg bw/d via diet.

Key result

Critical effects observed:

no

Reproductive performance:

no effects observed

Description (incidence and severity):

No treatment-related effect was observed for any of the indices. Also, EG treatment did not affect neonatal body weight at days 4, 14, or 21 post partum.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related histopathologic findings in F2 parents or in F3 weanlings. Although the kidney has been shown to be the primary target organ for EG-induced toxicity, there was no increase in the incidence or severity of kidney lesions in this study. One high dose F2 animal of each sex had mild focal interstitial nephritis. However, this condition was also seen in a control male and a control female. Unilateral hydronephrosis occurred in another high-dose F2 male. In addition, mild focal tubular hyperplasia was observed in one high-dose male F3 pup but was also diagnosed in two control male pups.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: there were no treatment-related effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reproductive Indices

		1.0 g/kg bw/d	0.2 g/kg bw/d	0.04 g/kg bw/d	0.0A	0.0B
F0 -> F1	Fertility index (%)	100	90	100	90	90
	Male	95	90	90	75	90
	Female	100	100	100	100	100
F1 -> F2	Fertility index (%)	100	100	90	90	90
	Male	85	95	85	90	85
	Female	100	100	100	100	94
F2 -> F3	Fertility index (%)	100	90	100	80	80
	Male	90	75	85	80	70
	Female	100	100	100	100	100

Neonatal body weight at day 21

		1.0 g/kg bw/d	0.2 g/kg bw/d	0.04 g/kg bw/d	0.0A	0.0B
F1 pups	males	30.6 +/- 4.5	30.9 +/- 4.9	30.7 +/- 6.4	30.6 +/- 3.6	27.9 +/- 4.3
	females	29.0 +/- 4.5	29.2 +/- 4.5	29.5 +/- 4.7	27.9 +/- 3.3	27.0 +/- 3.5
F2 pups	males	32.8 +/- 3.5	30.9 +/- 5.8	29.3 +/- 4.7	30.0 +/- 4.0	28.8 +/- 4.3
	females	30.8 +/- 3.4	30.2 +/- 4.9	28.8 +/- 3.8	28.5 +/- 3.1	27.5 +/- 3.4
F3 pups	males	30.2 +/- 4.0	30.9 +/- 4.0	30.9 +/- 4.0	32.0 +/- 3.9	30.2 +/- 4.6
	females	28.6 +/- 3.8	28.2 +/- 3.4	29.7 +/- 4.0	30.1 +/- 3.5	27.7 +/- 3.9

Conclusions:

In conclusion, there were no reproductive effects associated with the inclusion of as much as 1000 mg/kg bw/d of test item in the diet.

Reference 9

Endpoint:

reproductive toxicity, other

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

"Fertility Assessment by Continuous Breeding (FACB)" assay.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age: CD-1 mice were purchased at 6 weeks of age
- Housing: Two animals were housed per cage in polycarbonate shoebox type cages with stainless steel wire bar lids. Cages were rotated (relative placement) at least once a week.
- Diet: Purina certified rodent chow animal diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 20 to 70%
- Photoperiod (hrs dark / hrs light): 14-hour light cycle

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

WATER PREPARATION
- Rate of preparation of water: fresh preparation once every 2 weeks
- Storage temperature of water: room temperature, under yellow light

VEHICLE
- distilled water

Details on mating procedure:

Animals were randomly paired within each treatment group. Treatment was initiated at 11 weeks of age and was continued for 18 weeks (1 week of premating, 14 weeks of cohabitation, and 3 weeks thereafter). During this period, the following parameters were evaluated: body weight, number of litters produced, number of live pups per litter, minimum number of dead pups per litter, group body weight of live pups (males and females recorded separately), percent of infertile pairs and abnormal pups and a brief description of the deformity, if any.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aliquots of various dosage formulations were sent to Midwest Research Institute (MRI), Kansas City, MO, for analysis at week 1 of Task 1 and weeks 1, 5, 11, and 17 of Task 2.

Duration of treatment / exposure:

Task 1: 14 consecutive days
Task 2: 18 weeks (1 week prior to cohabitation, 14 weeks of cohabitation, and 3 weeks thereafter)
Task 4: 10 weeks

Frequency of treatment:

dosed/undosed water ad libitum, daily

Details on study schedule:

Fertility Assessment by Continuous Breeding (FACB). It consists of four related tasks, not all of which are necessarily performed for a given compound. These tasks include Task 1 - dose finding; Task 2 - cohabitation phase; Task 3 - identification of the affected sex and Task 4 - offspring assessment. This test protocol is designed to provide an alternative to multigeneration studies which produce similar comprehensive reproductive data but in a considerably shorter time and at a lower cost.
Task 1 is conducted to determine suitable doses for the continuous breeding phase. The test chemical is administered for 14 consecutive days and them maximum tolerated dose (MTD) is computed. Task 2 is designed to determine the effect of the MTD and two lower dose levels on fertility and reproduction. In this phase, treatment is continued for 18 weeks (1 week prior to cohabitation, 14 weeks of cohabitation, and 3 weeks thereafter). If the fertility is significantly affected, Task 3 is conducted to determine whether the male, female or both sexes are affected. If the overall response in Task 2 is negative, Task 4 is conducted. It is designed to evaluate reproductive performance in the offspring from the final and generally the fifth litter of the control and high dose groups. If the fertility in the first generation offspring is significantly affected, a Task 3 may be performed using these animals to determine the affected sex. At the conclusion of either Task 3 or 4, experimental animals may be necropsied.
During necropsy the liver, brain, pituitary, female reproductive tract (ovaries, oviduct, uterus, and vagina), testes, epididymis, prostate, and seminal vesicles with coagulating glands are weighed and fixed for histopathology. Based on the overall response during Task 3 or 4, vaginal smears are prepared to check the effect on oestrous cycle and sperm studied in detail to evaluate the effect on sperm density, sperm motility, and sperm head morphology.

Dose / conc.:

0 other: % in water (w/v)

Remarks:

dose range-finding, main study

Dose / conc.:

0.25 other: % in water (w/v)

Remarks:

dose range-finding, main study; corresponding to a dose of approx. 410 mg/kg bw

Dose / conc.:

0.5 other: % in water (w/v)

Remarks:

dose range-finding, main study; corresponding to a dose of approx. 840 mg/kg bw

Dose / conc.:

1 other: % in water (w/v)

Remarks:

dose range-finding, main study; corresponding to a dose of approx. 1640 mg/kg bw

Dose / conc.:

2.5 other: % in water (w/v)

Remarks:

dose range-finding

Dose / conc.:

5 other: % in water (w/v)

Remarks:

dose range-finding

No. of animals per sex per dose:

Task 1: 8/8 male/female
Task 2: 20/20 male/female treatment; 40/40 male/female vehicle control
Task 4: 20/20 male/female treatment and control

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the information available in the literature, the following dose levels were selected: 0, 0.25, 0.5, 1.0, 2.5, and 5.0% administered in drinking water.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- time schedule: twice daily

BODY WEIGHT: Yes
- time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- time schedule for examintations: weekly
- estimate of substance intake by multiplying water consumption by chemical concentration divided by the sum weight of the pair

Litter observations:

The following parameters were examined in [F1 / F2 / F3] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals, after litter was weaned

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Postmortem examinations (offspring):

SACRIFICE
- the F1 offspring was sacrificed after mating trial was completed
- these animals were subjected to postmortem examinsations as follows:
body weight,
organ weight: liver, brain, pituitary
reproductive tract: females: cranial half of the vagina, cervix, uterus, and ovaries; males: testes, epididymis, seminal vesicles and prostate

Statistics:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

Dose range-finding study: severe respiratory distress; at 2.5 and 5 % treatment groups
Main study: no clinical signy in all treatment groups

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Dose range-finding study: treament related deaths in the 2.5 (2/8 males) and 5 % (3/8 males; 1/8 females) groups
Main study: 2 females died in 0.5% treatment group; 1 males and 2 females died in control group

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Dose range-finding study: dilated renal tubules, tubular nephrosis related to oxalate crystal accumulation at 2.5 and 5 % treatment groups
Main study: moderate number of oxalate crystals in renal tubuli of one animal (0.5% treatment group)

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Main study: statistically significant decrease in number of litters per fertile pair, mean number of live pups, and mean live pup weight in 1 % treatment group. As well as significant reduced average total litter size; average number of male pups per litter; and both male and female pup weights.

Key result

Dose descriptor:

NOEL

Effect level:

0.5 other: percent in drinking water (w/v)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: equivalent to approx. 840 mg/kg bw/d

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

1 % treatment group: facial defects, pattern of skeletal defects, affected skull, cleft lip, abnormally shaped or missing sternbrae, fused ribs and abnormally shaped vertebrae, twisting of spine.
Neither the 0.25 nor 0.5% dose groups were significantly affected.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Exposure to ethylene glycol resulted in a small but significant decrease in the number of litters per breeding pair, in the number of live pups per pair and in the live pup weight. A significant number of pups in the 1.0% dose group were born with distinct facial deformities. In the retained litters at this dose, the facial deformities were more obvious with age. These malformed animals also exhibited fused ribs and shortened nasal, parietal, and/or frontal bones of the skull. When pups from the high dose group were raised to adulthood (with continued exposure to ethylene glycol) and mated, they exhibited decreased mating and fertility indices relative to controls handled in the same manner, but there were no effects on litter size, pup weight or sex ratio.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

0.5 other: percent in drinking water (w/v)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Remarks on result:

other: equivalent to approx. 840 mg/kg bw/d

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 other: percent in drinking water (w/v); equivalent to approx. 1640 mg/kg bw/d

System:

musculoskeletal system

Organ:

other: facial deformities

Treatment related:

yes

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

gross malformations; eight animals with distinct facial deformities in the 1% EG group were not necropsied and later utilized for skeletal examinations.

Skeletal Examination: A series of skeletal deformities were apparent in offspring delivered by the high dose (1% EG) group. Briefly, (a) frontal and nasal passages were significantly shortened and sometimes curved; (b) one or more pairs of ribs were fused; (c) one or more ribs were branched; (d) one or more centra were abnormal; and (e) parietals were smaller than the normal width. None of these abnormalities were noted in the untreated CD-1 mice.

Histopathological findings:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

facial and skeletal defects

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Fertility was 80% for the control group compared to 61% to those animals receiving 1% EG, but the difference was not statistically significant. The number of live pups per litter and the live pup weight were lower in the EG-treated group, as they were in task 2, but differences were not statistically significant in either case. Although no clinical signs of EG toxicity were reported in the study, unusual facial features were noticed in some of the offspring of the treated mice but not in the controls. The affected offspring generally had a shorter snout with wide-set eyes compared to the control CD- 1 mice. The examination revealed a pattern of skeletal defects in the treated mice affecting the skull, sternebrae, ribs, and vertebrae in both males and females. The defects included shortened frontal, nasal, and parietal bones; one or more pairs of fused ribs; abnormally shaped or missing sternebrae; abnormally shaped vertebrae; and twisting of the spine. The untreated mice showed no such defects. It was apparent from low-magnification examination of the histological sections that the size and shape of the bones in treated mice differed from the controls. Bones from treated mice were smaller and had altered shapes. However, histologic alterations were not evident when bones from treated mice were examined by light microscopy. Formation of lamellar bone, numbers and activity of osteoblasts and osteoclasts, and cartilage structure were identical in treated and control tissues. Tooth structure was normal in all treated mice. No alterations were seen in nasal turbinates, skeletal muscle, various salivary glands, eyes, or in those portions of the olfactory lobes of the brain which were frequently present in the sections. Deposition of calcium oxalate crystals were not found during careful examination of the microvasculature of bony or adjacent soft tissues. No other treatment-related effects were noted in the EG treated mice.

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

0.5 other: percent in drinking water (w/v)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skeletal deformities

Remarks on result:

other: equivalent to approx. 840 mg/kg bw/d

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 other: percent in drinking water (w/v); equivalent to approx. 1640 mg/kg bw/d

System:

other: facial deformities

Organ:

other: gross malformations

Treatment related:

yes

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 other: percent in drinking water (w/v); equivalent to approx. 1640 mg/kg bw/d

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

TASK 1:

EG administered in drinking water for 2 weeks at 0.25, 0.5, 1.0, and 2.5% (w/v) dose levels (equivalent to approx. 410, 840, and 1640 mg/kg bw/d) had no significant effect on body weights of both male and female mice. Mice exposed to 5% EG lost weight. The group mean body weights for control male mice at the beginning and the end of Task 1 were 34.4 and 34.6 g, respectively. Corresponding values for the 5% EG group were 34.9 and 31.4 g.

Certain animals in the 2.5 and 5% EG groups were sluggish and appeared to have respiratory problems within one week of treatment. Hair coat changed from normal to rough. During the later part of the first week or the early part of the second week, a number of animals in these two dose groups were lethargic and hunched. A significant number of these mice died. No such symptoms were noted in the control group or in animals in the 0.25, 0.5, and 1% EG groups.

Two of the eight male mice exposed to 2.5% EG died during Task 1. None of the female mice died in this dose group. One female and three male mice died in the 5% EG group. These animals exhibited severe respiratory distress prior to death. Pathologic examination showed distinct lesions in the kidneys and dilated tubule with moderate numbers of oxalate crystals in the lumina. The lungs were moderately congested. The cause of death was diagnosed as tubular nephrosis due to oxalate crystals.

Daily consumption of distilled water by the control mice or dosed water by the animals in the different treatment groups was essentially the same except for the male mice in the 5% EG group (p<0 .001).

 

TASK 2:

EG administered in drinking water at 0.25, 0.5, and 1% (w/v) dose level (equivalent to approx. 410, 840, and 1640 mg/kg bw/d) had no apparent effect on male or female body weights. The group mean body weights of male mice in the control and different treatment groups varied between 36.8 to 37.3 g at the beginning of Task 2. After 18 weeks of treatment, the group mean body weights for animals exposed to 0, 0.25, 0.5, and 1% EG were 40.3, 40.6, 40.2, and 40.3 g, respectively. Body weights for female mice varied considerably during Task 2 and the weight depended on the gestation phase.

Only five (5) mice died during 18 weeks of Task 2; one male and two females in the control group and two females in the 0.5% EG group. The cause of death for the control male mouse was mild myodegeneration of the heart and pulmonary congestion. One of the two control female mice revealed gravid uterus. Only one of the two female mice in the 0.5% EG group was necropsied. Pathologic examination revealed dilated tubule with moderate numbers of oxalate crystals in the lumina. The cause of death was suspected due to tubular nephrosis due to oxalate crystals.

The presence of ethylene glycol at a concentration of 1% or less did not significantly interfere with daily water consumption during Task 2 for both male and female mice.

Reproductive Performance and Fertility
Ethylene glycol administered continuously in drinking water at 0.25, 0 .5, and 1% dose levels had no effect on fertility in CD-1 mice. The fertility index for control and all three dose levels was 100%, i.e. every experimental pair delivered at least one litter. EG treatment at 1% dose level (approx. 1640 mg/kg bw/d) significantly reduced (p <0.05): (1) the average number of litters, 4.45 vs. 4.89 in the control group; (2) the average total litter size; (3) the average number of male pups per litter; and (4) both male and female pup weights. This is regarded as a sequel of developmental toxicity. No significant (p >0.05) differences existed between the 1% EG and the control group with respect to: (1 ) the proportion of live pups; and (2) sex ratio. EG treatment at 0.25 and 0.5% dose levels did not significantly (p >0.05) affect reproductive performace of CD-1 mice with respect to any of the above parameters. Interestingly, a significant number of pups delivered by breeding pairs in the 1% EG group showed distinct facial deformities. Six pups representing three different litters revealed a possible cleft lip/palate.

Gestation Period: EG exposure had no apparent effect on the gestation period. Cumulative days to 1st, 2nd, 3rd, 4th, and 5th litters were 28, 51, 69, 91, and 112 (days of the study), respectively. Corresponding values for the 1% EG group were 30, 55, 75, 96, and 111, respectively. It must be added that these values (days of the study) include 7 days of the premating period.

TASK 4:

The average pup weight at weaning ranged between 14.0 to 18.4 g. Offspring from both the control and 1% EG (approx. 1640 mg/kg bw/d) groups gained weight at essentially the same rate.

At least four male and four female mice among the offspring selected for Task 4 matings showed distinct facial deformities. These mice were later used for detailed skeletal examinations.

There was no mortality among the first generation offspring in the control group. Three offspring died in the 1% EG group; one male and two females. The cause of death was not treatment related.

Water consumption by first generation offspring from the control and 1% EG group was monitored on a weekly basis. There were no apparent difference in the average intake of distilled/dosed water by the control/1% EG group offspring.

Reproductive Performance and Fertility:
Twenty (20) pairs of first generation offspring were randomly selected from the control as well as 1% EG groups to assess their reproductive performance. The breeding pairs were mated until a copulatory plug was detected or for a maximum of seven days. The percent of plug positive/No. cohabited (mating index) for the control and treated pups was 90 and 74, respectively. Fertility was also affected by EG treatment. The percent of No. fertile/No. cohabited (fertility index) in the control and EG treated pups was 80 and 61, respectively. Other reproductive parameters were not significantly different (p>0.05) from the control values, i.e. the number of live pups per litter (males, females, or combined), proportion of pups born alive, and sex ratio.

Gross Necropsy:

No significant differences existed in terms of body weight, reproductive tract, and pituitary weight (p>0.05). Average brain weight of treated animals was lower than the control value (p<0.05). Organ weights were then adjusted for body weight by analysis of covariance. Male offspring body and organ weights for control and the treated animals were essentially the same except for the brain and right cauda. The brain weight was decreased by approximately 8 percent of the control value and right cauda weight by 14 percent. Male organ weights were also adjusted for body weight by analysis of covariance. It must be added here that eight animals with distinct facial deformities in the 1% EG group were not necropsied and later utilized for skeletal examinations.

Skeletal Examination:

A series of skeletal deformities were apparent in offspring delivered by the high dose (1% EG) group. Briefly, (a) frontal and nasal passages were significantly shortened and sometimes curved; (b) one or more pairs of ribs were fused; (c) one or more ribs were branched; (d) one or more centra were abnormal; and (e) parietals were smaller than the normal width. None of these abnormalities were noted in the untreated CD-1 mice.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

269 mg/m³

Study duration:

subchronic

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Fertility:

A study according to OECD TG 422 for HEA is available for this endpoint.

In a study according to OECD 422, HEA was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 12, 40 and 120 mg/kg body weight/day (mg/kg bw/day, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity (BASF SE, 2020). Control animals were dosed daily with the vehicle water. The duration of treatment covered a 5 weeks in-life period (males) including 14 days mating (mating pairs were from the same test group) as well as a 2-weeks premating period (females), 14 days mating period, 9 days post-mating period in one female (for no evidence of sperm), the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals. Regarding clinical examination, many male and female animals of the mid-dose group and all male and female animals of the high-dose group (40 and 120 mg/kg bw/day) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is likely, that this temporary finding was induced by a local affection of the upper digestive tract. Although salivation is not an adverse finding per se, in the present study it most likely resulted from the considerable irritating potential of the test item which caused erosions/ulcerations in the stomach of a number of mid- and high-dose animals.

Otherwise the test item caused no clinical signs of toxicity. Particularly, in the in-depth investigations including the detailed clinical observation (DCO), the functional observational battery (FOB) and the measurement of motor activity (MA) no treatment-related differences to control were observed at any dose level.

Food consumption was generally comparable to the concurrent control in all treated groups, except for the high-dose female animals throughout lactation. Overall the high-dose females consumed about 14% less food than the control females during this study section.

Mean body weights were generally not influenced by the treatment. Body weight gain was lower in all treated groups at the beginning of exposure (though statistically significant only in the high-dose parental males during study days 0 – 7), suggesting that the animals needed a few days to get adapted to the bolus gavage of this irritating compound. After this adaptation the mean body weight change was generally comparable between concurrent control and treatment groups during the remaining study.

Concerning clinical pathology and thyroid hormone values, no treatment-related, adverse effects were observed up to a dose of the compound of 120 mg/kg bw/day.

Regarding pathology, the target organ was the stomach. Mild to moderate squamous hyperplasia and erosions and/or ulcerations were found in the forestomach of males of the mid-dose group (40 mg/kg bw/day) and animals of both sexes of the high-dose group (120 mg/kg bw/day), correlating with the thickened Margo plicatus, the thickened wall and the foci found in gross pathology. Additionally, a submucosal edema with inflammatory infiltrates was noted in animals of test group 3. In the glandular stomach a minimal to mild hyperemia/edema in the lamina propria often accompanied by an attenuation of the overlying epithelial layer was noted in animals of both sexes of test group 3. When the above described findings in the glandular stomach and the forestomach coincided, they were regarded as treatment-related and adverse local effects. In the glandular stomach of some males and females also erosions and ulcerations were noted, without showing a dose-dependency, but correlating with macroscopic findings. Since there was no dose response and animals affected in test group 1 and 2 did not display the above described related histological lesions in the forestomach and glandular stomach at large, this finding is regarded as a spontaneous background lesion and not treatment-related.

All other pathology findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. Furthermore, neither the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina nor the stages of seminiferous tubules were altered.

Although the impact of the irritating potential of the test item was detected as local effects, one of the distinguished downstream consequences of the observed gastrointestinal pathology is pain, causing considerable distress and impairment of general condition in the affected animals. Collectively, salivation and gastrointestinal pathology indicate distinct parental toxicity in the high-dose animals and mild parental toxicity in the mid-dose animals.

Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups. F0 parental animals across all test groups proved to be fertile. Mating behavior, conception, implantation and parturition were not affected.

Overall, pre-postnatal development was not influenced by the test item, as there were neither significant changes of pup survival or well-being nor pup body weight/body weight gain. However, there were two findings which require more in-depth consideration.

The statistically significantly shifted in sex ratio in the high-dose group on PND 0 (males 62.8*[*p<=0.05] versus [vs.] 43.8 in control and females 37.2* vs. 56.2 in control) was slightly outside the historical control range (HCD: 36.6% - 60.5% (males), 41.5% - 63.4% (females)). Nevertheless, it was assessed as not treatment-related, because pup necropsy at PND 13 revealed no morphological evidence for virilization and none of the other endocrine-sensitive parameters (e.g. pup weight, AGD, nipple retention) gave any indication of a test substance-related effect in any of the dose levels (see below). Thus, these values were most likely outliers and considered to be spontaneous in nature and not treatment related.

The apparently lower pup survival rate (83.6% vs. 99.1% in control, statistically non-significant) in the high-dose group was caused by the two individual dams 132 and 138. Dam 132 had a large litter (14 pups) and was not able to nurse all her pups properly. Consequentially 5 pups died from undernutrition within 3 days after they were born. Histopathology revealed a liver torsion in this animal, which presumably has essentially contributed to this insufficient nursing behavior. Dam 138 had only one pup, which also died from undernutrition 3 days after it was born, thus contributing to the lower pup survival percentage in a disproportionate fashion. This single pup death was most likely an incidental event. Thus, the pup losses for both dams were considered to be unrelated to treatment.

Neither determination of anogenital distance/index nor the count of nipple/areola anlagen revealed any treatment-related changes up to and including a dose level of the test item of 120 mg/kg bw/d.

In conclusion, the oral administration of HEA by gavage to male and female Wistar rats resulted in signs of parental toxicity at the mid- and high-dose of 40 and 120 mg/kg bw/day, such as a combination of clinical signs and gastrointestinal pathology, being the consequence of the irritating properties of the test item. Thus, the no observed adverse effect level (NOAEL) for local toxicity was 12 mg/kg bw/day and  for general systemic toxicity 40 mg/kg bw/day for male and female Wistar rats. The NOAEL for reproductive performance and fertility was set to 120 mg/kg bw/day for male and female Wistar rats. The NOAEL for developmental toxicity was 120 mg/kg bw/day.

 

Hydroxypropyl acrylate: 

For the source substance HPA an OECD422 study (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test) is available in which no adverse effects on fertility were observed up to the 150 mg/kg bw/day, the highest dose tested (BASF 2018). The NOAEL for fertility and reproductive performance was 150 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 progeny was 150 mg/kg bw/d. HPA was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 15, 50 and 150 mg/kg body weight/day (mg/kg bw/day). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle water. The duration of treatment covered a 2-week premating and a mating period for both sexes, approximately 2 days post-mating in males, as well as gestation and lactation in females. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before the start of the administration period and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for GD 0-7, 7-14, 14-20 and PND 1-4, 4-7, 7-10 and 10-13. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. Anogenital distance measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement. Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. At the 150 mg/kg bw/day dose group the following observations were recorded: decreased food consumption in the females during the entire premating period (up to 7% below control), minimal to slight thickening of the mucosa of the duodenum correlating to the macroscopically observed dilation in all male and 9/10 females, focal hyperplasia of the duodenal mucosa in 1/10 female animals, diffuse squamous hyperplasia of the forestomach in all male animals (graded slight or moderate) and in 7/10 female animals (graded minimal or moderate), erosion/ulcer in the cranial part of the forestomach in 6/10 male and 2/10 female animals. In the mid dose group minimal thickening of the wall of the duodenum correlating to the macroscopically observed dilation in 2/10 male animals and minimal diffuse squamous hyperplasia of the forestomach in 6/10 male and female animals. No observations in the low dose-group were reported. In all dose groups no test substance-related adverse findings were reported for the F1 animals. The NOAEL for general, systemic toxicity of HPA was 150 mg/kg bw/day for male and female rats. Based on pathological findings in forestomach and duodenum in F0 parental rats of both sexes at 150 and 50 mg/kg as well as corresponding reductions of food consumption in F0 females at 50 mg/kg bw/day a NOAEL of 15 mg/kg bw/day was determined for local effects in the gastrointestinal tract. The NOAEL for fertility and reproductive performance was 150 mg/kg bw/day for the F0 parental rats. The NOAEL for developmental toxicity in the F1 progeny was 150 mg/kg bw/day.

Hydroxypropyl acrylate (HPA) was examined in a in the Extended One Generation Reproductive Toxicity Study according to OECD TG 443. HPA was administered to groups of 25 male and 25 female healthy young Wistar rats as test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 15, 50 and 150 mg/kg body weight/day [mg/kg bw/d]). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group.Pups of the F1 litter were selected (F1 rearing animals) and assigned to 3 different cohorts (1A, 1B and 3) which were subjected to specific postweaning examinations.The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Control animals were dosed daily with the vehicle (drinking water).

A detailed clinical observation (DCO) was performed in all F0 parents and F1 animals in cohorts 1A, 1B, 2A and 3 at weekly intervals.

Estrous cycle data were evaluated for F0 females over a three weeks period prior to mating until evidence of mating occurred.In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75. Moreover, the estrous stage of each F0, 1A and 1B female was determined on the day of scheduled sacrifice.

The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically.

Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 pups selected to become F1 rearing animals was recorded.

All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one PND 20. The number of nipple/areola anlagen were counted.

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live F1 pups on PND 1.

Urine samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group.

Blood samples for clinical pathological investigations were withdrawn from 10 F0 parental and cohort 1A animals per sex and group.

Furthermore, blood samples were taken from all surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group.

Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 and 1A males at scheduled sacrifice or after appropriate staining.

All F0 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females of cohort 1A. All F1 rearing animals were assessed by pathological examinations.

150 mg/kg bw/d

F0 Parental animals: Salivation after gavage dosing in almost all male and female animals across all study periods.Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values in females. Increased absolute reticulocyte counts in females. Increased absolute and relative neutrophil cell counts in males. Decreased relative lymphocyte counts in males. Gross finding “focus” in the forestomach of 18 male and 6 female animals. Gross finding “margo plicatus thickened” in the forestomach 17 males and 25 females. Gross finding “focus” in the glandular stomach of 4 male and 7 female animals. Erosion/ulcer in the forestomach in 19 males and 6 females, correlating to the gross finding “focus”. Hyperplasia of the squamous cell layer of the margo plicatus in the forestomach of 16 males (graded up to moderate) and 24 females (graded up to marked), correlating to the gross finding “margo plicatus thickened”. Hyperplasia squamous cell diffuse in the forestomach in 6 males and 11 females, mostly graded minimal. Multifocal degeneration/regeneration of the glandular stomach in 15 male and 12 female animals, mostly graded minimal to slight. Erosion/ulcer in the glandular stomach in 6 males and 12 females.

F1 pups: No test substance-related adverse findings

F1 rearing animals, cohort 1A: Salivation after gavage dosing in all male and female animals during the entire study.Increased urea and inorganic phosphate levels in females.Gross finding “focus” in the forestomach of 6 male and 3 female animals. Gross finding “margo plicatus thickened” in the forestomach of 20 males and 19 females. Erosion/ulcer in the forestomach of 6 males and 4 females, correlating to the gross finding “focus”. Hyperplasia of the squamous cell layer of the margo plicatus in 20 males and 18 females graded up to moderate in males and slight in females, correlating to the gross finding “margo plicatus thickened”. Minimal diffuse hyperplasia squamous cell in the forestomach of 6 male animals. Mostly minimal multifocal degeneration/regeneration in the glandular stomach of 7 male and 3 female animals.

F1 rearing animals, cohort 1B:Salivation after gavage dosing in nearly all male and all female animals during the entire study.Gross finding “focus” in the forestomach of 4 male and 3 female animals. Gross finding “margo plicatus thickened” in the forestomach of 24 males and 22 females.

F1 rearing animals, cohort 3: Salivation after gavage dosing in all male and female animals during the entire study.

50 mg/kg bw/d

F0 parental animals: Salivation after gavage dosing in about half of the animals across all study periods.Gross finding “focus” in the glandular stomach of 2 male and 12 female animals. Gross finding “margo plicatus thickened” in the forestomach of 24 males and 22 females. Erosion/ulcer in the glandular stomach of 6 males and 12 females, correlating to the gross finding “focus”. Minimal hyperplasia of the squamous cell layer of the margo plicatus in the forestomach of two males and three females, correlating to the gross finding “margo plicatus thickened”.

F1 pups: No test substance-related adverse findings

F1 rearing animals, cohort 1A:Salivation after gavage dosing in many male and female animals during the entire study.Gross finding “margo plicatus thickened” in the forestomach of 6 males and 2 females. Hyperplasia of the squamous cell layer of the margo plicatus in the forestomach of 6 males and 3 females graded minimal, correlating to the gross finding “margo plicatus thickened”. Hyperplasia squamous cell diffuse in the forestomach of one male animal.

F1 rearing animals, cohort 1B: Salivation after gavage dosing in many male and female animals during the entire study. Gross finding “focus” in the forestomach of 1 female animal. Gross finding “margo plicatus thickened” in the forestomach of 2 females.

F1 rearing animals, cohort 3: Salivation after gavage dosing in many male and female animals during the entire study.

15 mg/kg bw/d

F0 parental animals: Gross finding “focus” in the glandular stomach of 3 male and 14 female animals. Erosion/ulcer in the glandular stomach of two males and 13 females.

F1 pups:No test substance-related adverse findings

F1 rearing animals, cohort 1A: No test substance-related adverse findings

F1 rearing animals, cohort 1B: No test substance-related adverse findings

F1 rearing animals, cohort 3: No test substance-related adverse findings

Under the conditions ofthe present extended 1-generation reproduction toxicity studythe NOAEL (no observed adverse effect level) for general, systemic toxicity is 50mg/kg bw/dfor the F0 parental as well as F1 adolescent animals, based on changed clinical-pathological parameters, which were observed at the LOAEL (Lowest Observed Adverse Effect Level) of 150 mg/kg bw/d. The NOEL (no observed effect level) for this study is below 15 mg/kg bw/d, based on clinical and pathological evidence of distinct local toxicity in the upper digestive tract, at all tested dose levels. These local effects were definitely dose-limiting.The NOAEL for fertility and reproductive performance for the F0 parental rats is150 mg/kg bw/d, the highest dose tested. The NOAEL for developmental toxicity in the F1 progeny is 150 mg/kg bw/d, the highest dose tested. The NOAEL for developmental immunotoxicity for the F1 progeny is150 mg/kg bw/d, the highest dose tested. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral) demonstrated that the test system worked properly (HARTF, 2023).

 

Methyl acrylate:

 

For the source substance MA a 2-generation study is available.In this study, according to OECD TG 416, groups of 27 male and female Crl: CD(SD) rats were whole-body exposed to MA vapours at target concentrations of 0, 5, 25, and 75 ppm for six hours/day, seven days/week, resulting in actual average concentrations of 0, 5.3 ± 0.2, 25.7 ± 0.3, and 75.4 ± 0.6 ppm, respectively (corresponding to approx. 0, 0.019, 0.092, and 0.269 mg/L). Rats were exposed daily for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation for two generations. Maternal rats were not exposed after GD 20 through LD 4 in order to allow for parturition and initiation of lactation. Exposure of maternal rats continued from LD 5 – LD 28. In-life parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In addition, post-mortem evaluations included gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.Treatment-related effects in parental rats exposed to 75 ppm included decreased body weight and feed consumption in males and females throughout most of the two generation study. There were no effects on body weight or feed consumption at 25 or 5 ppm. Treatment-related, adverse histopathologic effects were present in the nasal tissues of P1 and P2 males and females exposed to 25 or 75 ppm. The incidence and severity of the nasal effects were concentration dependent. Degeneration with regeneration of the olfactory epithelium (very slight to moderate) occurred in all P1 and P2 males and females exposed to 75 ppm. Very slight olfactory epithelial degeneration, without accompanying regenerative hyperplasia, was noted in some of the P1 and P2 females and P2 males exposed to 25 ppm. There were several histopathologic effects that accompanied the degeneration of the olfactory epithelium. Very slight or slight degeneration of the olfactory nerve was present in most of the P1 and P2 males and females exposed to 75 ppm, and one P1 male exposed to 25 ppm. Very slight or slight chronic-active inflammation was present in 16/27 P1 males, 20/27 P1 females, 14/27 P2 males, and 8/27 P2 females exposed to 75 ppm, and in one or two males and females from both generations exposed to 25 ppm. Very slight necrosis of individual olfactory epithelial cells was present in most of the P1 and P2 males and females exposed to 75 ppm, and a few P1 and P2 animals (one to four per sex) exposed to 25 ppm. Very slight mineralization of the olfactory epithelium was present in one or two P1 and P2 animals exposed to 25 ppm, and in 6/27 P1 males, 4/27 P1 females, 16/27 P2 males and 14/27 P2 females exposed to 75 ppm. Other nasal effects consisted of an increase in the incidence of very slight or slight hyperplasia of the transitional epithelium in P1 and P2 males and females exposed to 25 or 75 ppm, and an increase in the incidence of very slight or slight hyperplasia and hypertrophy of the respiratory epithelium in P1 males and females exposed to 25 or 75 ppm, and in P2 males and females exposed to 75 ppm. There were no treatment-related histopathologic effects in P1 or P2 animals exposed to 5 ppm.No treatment-related effects were seen in reproductive function or pup survival. However, pup body weights of the 75 ppm exposure group were decreased on postnatal day 14-28 in both generations. There were no effects on pup body weight in rats exposed to 25 or 5 ppm. The effects on pup body weight, as well as the changes in parental body weight and feed consumption, likely were secondary changes all stemming from nasal irritation and resultant stress.In summary, the no-observed-adverse-effect concentration (NOAEC) for parental systemic toxicity was determined to be 5 ppm (= ca. 0.018 mg/L) and was based on histologic changes in the nasal tissues seen at higher concentrations. The NOAEC for developmental toxicity was 25 ppm (= ca. 0.089 mg/L), based on decreases in pup body weight at 75 ppm which were secondary to parental toxicity. The NOAEC for reproductive toxicity was 75 ppm (= ca. 0.268 mg/L), the highest concentration tested (Dow. Chem. co. 2009).

 

n-Butyl acrylate:

 

For the source substance nBA an Extended One Generation test (OECD443) is available. In this study30 Crl:CD(SD) rats were exposed to 20, 50 and 150 mg/kg bw/day by oral (gavage) exposure route.

There were no test substance-related effects on survival for F0 and F1 animals at any dosage level. No test substance-related clinical observations were noted for F0 and F1 animals at any dosage level. Mean body weights, body weight gains, food consumption, and food efficiency in the 20, 50, and 150 mg/kg/day F0 and F1 males and females were unaffected by test substance administration. No test substance-related effects were noted on F0 reproductive performance (male and female mating and fertility, male copulation, and female conception indices), the mean number of days between pairing and coitus, mean gestation lengths, or the process of parturition. In addition, there were no test substance-related effects on F0 or F1 estrous cyclicity or spermatogenic parameters (testicular and epidydimal sperm concentrations, sperm production rate, sperm motility, and sperm morphology) at any dosage level.

There were no test substance-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 postnatal survival, clinical observations, anogenital distance,

offspring body weights, necropsy findings, or developmental landmarks (areolae/nipple retention, vaginal patency, and balanopreputial separation).

No test substance-related effects on clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were noted for F0 and F1 animals at any dosage level. In addition, no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) were noted in F0 and F1 males or females or F1 pups (on PND 4 and 21). Test substance-related histologic changes were observed in all dosage groups in the F0 generation and F1 males and females in Cohort 1A. Epithelial hyperplasia and/or hyperkeratosis was observed in the nonglandular stomach in all test substance-treated groups examined. Mild to moderate changes in the 150 mg/kg/day group males and females of the F0 and F1 generations were considered adverse in this study. Microscopic changes in the stomach were associated with the gross observation of thickened nonglandular stomach, but were not associated with any clinical pathology, organ, or body weight changes. In the F0 generation, a nonadverse increased incidence of biliary hyperplasia (males and females) and random hepatocellular necrosis (males) were observed in the liver in the 150 mg/kg/day group. Additionally, nonadverse test substance-related microscopic findings (increased severity of mineralization at the corticomedullary junction) were observed in the kidneys of the 150 mg/kg/day group F0 females. Thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females at the scheduled necropsies for Cohort 1B; this finding was considered test substance-related and adverse in the 150 mg/kg/day group males and females. No other test substance-related internal findings were observed at any dosage level for F1 Cohort 1B animals. No test substance-related effects on the mean number of F0 implantation sites or number of unaccounted-for sites were noted at any dosage level. No test substance-related macroscopic findings were observed in F1 pups that were found dead, culled on PND 4, or examined at the scheduled necropsy on PND 21; F1 pup organ weights on PND 21 were unaffected by test substance administration. No test substance-related effects on ovarian primordial follicle counts were noted in the F0 females suspected of reduced fertility or F1 Cohort 1A females. There were no test substance-related effects on organ weights noted for F0 and F1 males and females at any dosage level.

Due to the absence of systemic toxicity noted for F0 and F1 males and females throughout the study, a dosage level of 150 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1 male and female systemic toxicity when nBA was administered orally by gavage to Crl:CD(SD) rats. Epithelial hyperplasia and/or hyperkeratosis in the nonglandular stomach noted in the 150 mg/kg/day group F0 and F1 males and females were considered adverse; based on these results, 50 mg/kg/day was considered to be the NOAEL and 150 mg/kg/day was considered to be the lowest-observed-adverse-effect level (LOAEL) for local effects in the F0 and F1 generations. Based on the lack of effects noted for F1 litters, a dosage level of 150 mg/kg/day was considered to be the NOAEL for neonatal toxicity. There was no evidence of reproductive toxicity at any dosage level based on evaluation of reproductive performance in the F0 generation and sperm measurements and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 150 mg/kg/day. (Charles River 2017)

In addition a subchronic inhalation study in rats demonstrated no indication of a fertility impairing potential of nBA (BASF 1979)

Acrylic Acid:

For AA possible effects on reproductive performance were investigated by oral administration (via drinking water) in two different studies with rats. From these studies it is concluded that the substance does not effect fertility:

In a one-generation study with F344 rats (Bushy Run Research Center, Union Carbide (1980b)), the animals (10 males and 20 females per dose group) received AA at dose levels corresponding to 0, 83, 250 or 750 mg/kg bw/d for 13 weeks. Each male was then mated with 2 females and exposure continued for both sexes throughout gestation and lactation. Dose-related reductions in food and water consumption and consequently in body weight gain were observed in the F0 animals, most pronounced and statistically significant at the 750 mg/kg bw/d dose level. In the high-dose group, pups of both sexes showed decreased body weight gain. Also in F0 males of the high-dose group a reduction in absolute and relative liver weights and in F0 females a reduction in both absolute and relative spleen weights was observed. At the high-dose level the fertility index of males and females, the gestation index, the number of pups born alive and the percentage of pups weaned were numerically, but not statistically significantly reduced. However, the data should be interpreted cautiously because of a relatively atypical control group, in which the female fertility index and the mean number of pups born alive/litter were reduced compared to the historical control of the testing laboratory.Therefore, based on the above findings the maximum dosage level that did not produce a deleterious reproductive effect for one generation of exposure of AA in the drinking water of F344 rats was estimated to be 250 mg/kg bw/day (= NOAEL for reproductive effects in the F0 and F1 generation).

In a two-generation study according to OECD TG 416 AA was administered orally in the drinking water to male and female Wistar rats at doses of 0, 500, 2500, 5000 ppm (corresponding to approx. 53, 240, 460 mg/kg bw/day). At least 70 days after the beginning of treatment, F0 animals were mated to produce one litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dosing group as their parents. Groups of 25 males and 25 females selected from F1 pups as F1 parental generation were offered drinking water containing 0, 500, 2500 and 5000 ppm of the test substance post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals.It can be concluded that the continuous administration of aqueous AA solutions to rats over two generations caused clear signs of toxicity in the highest dose group (5000 ppm =approx. 460 mg/kg body weight/day) in F0 and F1 parents. General toxicity was substantiated by e. g. reduced food and/or water consumption, impairment of body weights/body weight gains and gross and histopathological findings in the fore- and the glandular stomach (i. e. thickening of and minimal hyperkeratosis at the limiting ridge (margo plicatus), edema in the submucosa of the glandular stomach), which are a consequence of the administration of the acid solutions (indicative of the irritating properties of the test substance). At 2500 ppm (= approx. 240 mg/kg body weight/day) the water consumption of the F1 parental animals was still clearly reduced, but no further substance-related adverse effects on the parental rats were seen.Clear adverse substance-induced effects were also noted for the progeny of the high dose of the F0 and F1. Impaired body weight/body weight gain in the F1 and F2 pups and some indications for delays in the morphological development of the F2 pups were seen. The latter finding was likely associated with the decreased body weight/body weight gain. Similar, but much less pronounced effects were also observed for the F1 and/or F2 pups at 2500 ppm.500 ppm (= approx. 53 mg/kg body weight/day) were tolerated by both parental generations and their offspring without any changes which could be causally related to test substance administration.AA had no adverse effects on reproductive parameters of the parental animals of either generation (F0 and F1) of all groups (500, 2500 and 5000 ppm). No adverse effects on fertility and pre-implantation development could be detected; no effects on reproductive organs have been observed. The mating index of males in both generations and in all dose groups was 100 %. The fertility rate in the F0 generation was between 92-96 %; in the F1 generation in all dose groups the fertility rate was 100 %. The rate of pregnancy in both generations was not reduced. In both generations there were no differences in numbers of pups born alive.Therefore, the NOAEL (no observed adverse effect level) with respect to reproductive function was 5000 ppm (=approx. 460 mg/kg body weight/day). The NOAEL with respect to general toxicity of the test substance was 2500 ppm (= approx.240 mg/kg body weight/day) for the F0 generation parental animals and 500 ppm (= approx. 53 mg/kg body weight/day) for the F1 males and females and the offspring (F1 and F2 pups).

 

Ethan -1,2-diol:

 

For the HEA metabolite ethane-1,2-diol a 3-generation study is available and a fertility assessment of continuous breeding are performed.

DePass et al. (1986) reported a three-generation reproduction toxicity study. Male and female rats were given orally (feed) daily doses of 40, 200 and 1000 mg/kg bw/d. No reproductive effects associated with the inclusion of as much as 1000 mg/kg bw/d of ethane-1,2-diolin the diet were found. The NOAEL for parental animals and for offsprings was found to be > 1000 mg/kg bw/d.

 

Gulati and Lamp (1984)reported a FACB study and found a NOEL of 840 mg/kg bw/d for parental and F1 male and female mice. Ethane-1,2-diol was administered in drinking water in concentrations of approx. 410, 840 and 1640 mg/kg bw/d. Exposure toethane-1,2-diolresulted in a small but significant decrease in the number of litters per breeding pair, in the number of live pups per pair and in the live pup weight. A significant number of pups in the 1.0% dose group (1640 mg/kg bw/d) were born with distinct facial deformities. In the retained litters at this dose, the facial deformities were more obvious with age. These malformed animals also exhibited fused ribs and shortened nasal, parietal, and/or frontal bones of the skull. When pups from the high dose group were raised to adulthood (with continued exposure to ethylene glycol) and mated, they exhibited decreased mating and fertility indices relative to controls handled in the same manner, but there were no effects on litter size, pup weight or sex ratio. These observations at high dosis seem to be rodent specific, recent investigation demonstrated that GA uptake into the rat embryo occurs predominantly by a specific, pH-dependent, active uptake transporter protein, consistent with the proton-linked monocarboxylate transporters (MCT)

Effects on developmental toxicity

Description of key information

No indications of a developmental toxic or teratogenic effect were seen in animal studies with 2-hydroxyethyl acrylate in rats and rabbits.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-05-2020 - 03-02-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Species and strain
Sexually mature, virgin New Zealand White rabbits (Crl:KBL(NZW)) were supplied at 15-18 weeks of age by Charles River Laboratories, Research Models and Services, Germany GmbH (Breeder: Charles River Laboratories, France). Only animals free from clinical signs of disease were used for the investigations.

Animal identification
The breeder produced unique identification of the rabbits by ear tattoo.

Reason for species selection
The strain was selected since historical control data is available from the test facility for New Zealand White rabbits. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

HOUSING AND DIET
During the acclimatization and study period, the rabbits were housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH, Hohenpeißenberg, Germany (floor space 4264 mm², internal height 450 mm).

For enrichment, wooden gnawing blocks were added (Typ SAFE® gnawing block), supplied by
J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany).

The animals were accommodated in fully air-conditioned rooms in which central air conditioning maintained a range of temperature of 17-21°C and a range of relative humidity of 45-65%. The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study.

The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h).

Before the study started, the animal room was completely disinfected using a disinfector (Geschko MLT 17 hydrogen peroxide gas generator (PEA; Germany)). The walls and the floor were cleaned once a week with water containing an appropriate disinfectant.

The food used was pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland. Food and drinking water (potable tap water in water bottles) were available ad libitum throughout the study (from the day of arrival to the day of necropsy).

Based on the pregnant animals the body weight on GD 0 varied between 3251 – 4432 g.

IN-LIFE DATES: From: 29 May 2020 To: 14 Jul 2020

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water over a period of a maximum of 7 days at room temperature had been verified prior to the start of the study with the same batch.

Samples of the test substance preparations were sent once (at the beginning of administration) to the analytical laboratory for verification of the concentrations.

All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology. All reserve samples and further samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C) Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to original samples after agreement by the Study Director.

The results of the analyses of the test substance preparations in drinking water confirmed the correctness of the prepared concentrations. The measured concentrations of the samples of treatment groups corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination.

A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.

During the acclimatization period the animals were assigned to the different test groups according to a randomization plan (NIJENHUIS and WILF) and on the basis of their body weights.

The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.

Duration of treatment / exposure:

The test substance was administered to the animal orally by gavage from implantation to one day prior to the expected day of parturition (GD 6-28) always at approximately the same time of day. The animals of the control group were treated in the same way with the vehicle (drinking water). The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

Frequency of treatment:

once daily

Dose / conc.:

6 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

On GD 29 blood samples were obtained in randomized order from all surviving animals from the ear veins. After blood sampling, the females were euthanized and examined. The fetuses were removed from the uterus and investigated.

Dose selection rationale
The high dose was selected based on signs of toxicity noted at dose levels of 90 and 180 mg/kg bw/d in a previously conducted maternal toxicity range-finding study (BASF project 20R0173/16R191, non-GLP) which preceded this definitive prenatal developmental toxicity study.
In this maternal toxicity range‐finding study, 5 presumed pregnant NZW rabbits were adminis- tered the test substance via oral gavage from gestational day (GD) 6 through GD 28, at doses of 90 and 180 mg/kg bw/d.
The 180 mg/kg bw/d dose caused in the pregnant rabbits an immediate cessation of food consumption after the first dosing. One animal was found dead and all remaining animals were killed for humane reasons after the second dosing.
The 90 mg/kg bw/d dose caused a significant reduction of food consumption (up to 61% below control) during the first two treatment weeks (GD 6-19). During the last treatment week recovery was noted, however, mean food consumption stayed about 14 % below control during the treatment period. In addition, clinical signs like poor general condition, high-stepping gait, blood in bedding and no or reduced defecation were observed in individual rabbits. One animal aborted.
Clinical pathology revealed no relevant findings.
At macroscopic pathology discolorations and foci were detected in the stomach of 3 rabbits at 180 mg/kg bw/d and one rabbit at 90 mg/kg bw/d. Histopathologically erosions/ulcers were detected in all 180 mg/kg bw/d rabbits and one 90 mg/kg bw/d rabbit. At 180 mg/kg bw/d these were accompanied by signs of minimal or mild to moderate hemorrhages and inflammation.
Thus, the selected high dose for the present definitive study (60 mg/kg bw/d) represented a dose which was supposed to cause a moderate reduction of food consumption and/or body weight/body weight gain in pregnant female rabbits. Considering the specific characteristic of rabbit gastrointestinal physiology and the consequences of their disruption for the maintenance of pregnancy in rabbits this procedure meets the principles of guidelines OECD 414 (adopted 2018) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in compliance with EU Directive 2010/63/EU on animal protection.

Maternal examinations:

Mortality
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).


Clinical symptoms
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.
During the administration period (GD 6-28) all animals were checked daily for any abnormal clinical signs before the administration as well as within 5 hours after the administration.


Food consumption
The consumption of food was recorded daily during GD 0-29.


Body weight data
All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.


Corrected (net) body weight gain
The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).


Clinical Pathology
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units.


The following examinations were carried out in all animals per test group:

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):

Parameters and methods:

Parameter Unit Method
Leukocyte count (WBC) giga/L cytochemistry coupled with flow cytometry
Erythrocyte count (RBC) tera/L flow cytometric laserlight scattering
Hemoglobin (HGB) mmol/L cyanmethemoglobin method; according to ICSH
Hematocrit (HCT) L/L calculation: MCV x erythrocytes
Mean corpuscular volume
(MCV) fL RBC/PLT method; mean of RBC volume distribution curve (histogram)
Mean corpuscular
hemoglobin (MCH) fmol calculation: hemoglobin/erythrocytes
Mean corpuscular
hemoglobin concentration
(MCHC) mmol/L calculation: hemoglobin/hematocrit
Platelet count (PLT) giga/L flow cytometric laserlight scattering
Differential blood count % & giga/L cytochemistry coupled with flow cytometry
Reticulocytes (RETA) giga/L cytochemistry coupled with flow cytometry


Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters
Parameters and methods:
See table below in any other information


Necropsy
On GD29 all surviving animals were sacrificed by an intravenous injection of pentobarbital (dose: 2ml/animal) in a randomized sequence. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
All animals which died intercurrently (animal No. 31) or were sacrificed in a moribund state (including animals with abortion; animal No. 91) were necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights were determined in all does sacrificed on schedule:
1. Adrenal glands
2. Kidneys
3. Liver
4. Spleen
All paired organs were weighed together (left and right).

The carcass weights (GROSSE-System) were transferred to the ACOPAT-System to calculate the relative organ weights. Organ weights of animals, that were not pregnant, were excluded from the calculation of mean values.

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Duodenum
4. Kidneys
5. Liver
6. Spleen
7. Stomach (forestomach and glandular stomach)

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the following table:

Organs Test groups
0 1 2 3
1. Stomach (forestomach and glandular stomach) A1 A1 A1 A1
2. Duodenum A1 A1 A1 A1
A = hematoxylin and eosin (H&E) stain 1 = all animals/test group

A correlation between gross lesions and histopathological findings was attempted.

Ovaries and uterine content:

Cesarean section

On GD 29, the surviving does were euthanized in randomized order by intravenous injection of pentobarbital (Narcoren®; dose 2 mL/animal). After exsanguination, the animals were necropsied and assessed by gross pathology.

The uteri and the ovaries were removed and the following data were recorded:

- Weight of the unopened uterus

- Number of corpora lutea

- Number and distribution of implantation sites classified as:

• Live fetuses

• Dead implantations:

a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)

b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)

c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the does (except of gross pathology including organ sampling and weights) and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.

Blood sampling:

In the morning blood was taken from the ear vein from not-fasted animals without anesthesia. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.


Additional blood samples for non-GLP research purposes

Additional samples during venipuncture were collected for external research projects beyond the scope of this study and without GLP status. The results of the study were not influenced by this procedure because blood sampling occurred just prior to sacrifice/at necropsy. The last aliquot was taken for these projects, thus blood sampling for the regulatory investigations was not affected. Thus, the sampling procedures did not affect the outcome and compliance of the GLP study. The data from these research projects do not affect the outcome, assessment and compliance of this GLP study.
From each animal 1 mL of blood was collected with EDTA as anticoagulant (10 µL of a 10% solution). The samples were centrifuged. The plasma was separated. The blood was sampled and prepared in original Eppendorf tubes. The preparation of the samples was done under cooling. All samples were stored covered with a N2 atmosphere and then stored at –80°C for research.

Fetal examinations:

All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

Examinations of the fetuses after dissection from the uterus
At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded.
Thereafter, the fetuses were sacrificed by an intraperitoneal injection of pentobarbital (Narcoren®; dose: 0.2 mL/fetus; one part Narcoren® diluted with one part physiological saline).

Soft tissue examination of the fetuses
After the fetuses had been sacrificed, the abdomen and thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure.
The sex of the fetuses was determined by examination of the gonads in situ.

After these examinations, the heads of approximately one half of the fetuses per doe (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN’s solution and were, after fixation, processed and evaluated according to WILSON’s method (WILSON and WARKANY). About 10 transverse sections were prepared per head. After the examination the heads were discarded.

All fetuses (including those without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the intact fetuses were removed from the fixative and a transversal incision was made into the frontal/parietal head bones. The two halves of the calvarium were cautiously bent outward and the brain was thoroughly examined. Subsequently, these fetuses were placed back into the fixative for further fixation.

Skeletal examination of the fetuses
After fixation in ethyl alcohol, the skeletons (with and without skulls) were stained according to a modified method of KIMMEL and TRAMMELL. The stained skeletons were placed on an illuminated plate, investigated and archived individually.

Evaluation criteria for assessing the fetuses
Classification and assessment of fetal findings is a matter of ongoing discussion (see e.g. BELTRAME and GIAVINI, CHAHOUD, SOLECKI). Despite considerable efforts to harmonize the nomenclature used to describe observations of fetal morphology, the terms still vary considerably between laboratories, investigators and textbooks in the fields of teratology and developmental toxicity.
In the present study the internationally harmonized glossary of WISE et al. (1997) and the updated version MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI:

Malformation
A permanent structural change that is likely to adversely affect survival or health.

Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.

All fetal findings were listed in tables according to these classifications.

Statistics:

see any other information below

Indices:

The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals / number of fertilized animals) x 100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of corpora lutea – number of implantations / number of corpora lutea) x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of implantations – number of live fetuses) / number of implantations) x 100

Historical control data:

see attached file

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No defecation was observed in three control females, one low-dose, two mid-dose and six high-dose females. Reduced defecation was observed in eight control, four low-dose, four mid- dose and seven high-dose females (0, 6, 20 and 60 mg/kg bw/d). Incidence and distribution of these findings do not indicate a relationship to the test substance.

There were no further clinical findings in the other does in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One low-dose female (No. 31 - 6 mg/kg bw/d) was found dead before treatment on GD 26, after showing no or reduced defecation during several days before. The cause of this preterminal death remains unknown, because of the lacking dose-response it is, however, considered not to be related to the test substance.

One high-dose female (No. 91 - 60 mg/kg bw/d) was sacrificed after abortion ahead of schedule (GD 25). Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, this was considered to be a spontaneous incident.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant difference was observed for the mean body weights (BW) of the high- dose females (60 mg/kg bw/d) when compared to the control group.

Collectively, the mean body weight gain during the treatment period was comparable between all test groups (6, 20 and 60 mg/kg bw/d) and the control group.

Notably, there were episodes of significant differences between control and high-dose group such as GD 19-21 where the high-dose does lost less weight (-3.9 g vs. -55.4 g in control), or GD 23-25 where they gained more weight than the control does (47.2 g vs. 6.8 g in control). These episodes compensated for a longer time period (GD 6-19) where the high-dose rabbit gained consistently (but not statistically significantly) less body weight than the controls.

Corrected (net) body weight gain
Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were not significantly different between all test groups including controls (0, 6, 20 or 60 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In comparison to the control group the mean food consumption of the does in test group 3 (60 mg/kg bw/d) was reduced from GD 12 onwards, attaining statistical significance on GD 12-14 and GD 15-18 (up to 40% below control). Afterwards it recovered, being comparable to or even exceeding the control values (attaining statistical significance on GD 25-27). Overall, the high- dose does consumed about 9% less food than the concurrent control does during the treatment period (GD 6-28).

The food consumption of the low- and mid-dose rabbits (6 and 20 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes of hematological parameters were observed.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

At gestation day 29, in does of test group 3 (60 mg/kg bw/d) cholesterol values were significantly increased, but the mean was within the historical control range (cholesterol 0.18-0.53 mmol/L). Therefore, this change was regarded as incidental and not treatment related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute weights
All mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative kidney weights were statistically significantly increased in test group 3 :

Relative weights
Test group (mg/kg bw/d) 1 (6) 2 (20) 3 (60)
Kidneys 96% 104% 110%**
*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Since only the relative kidney weights in test group 3 were significantly increased and since relative kidney weights lay within the range of historical control values, weight changes were regarded as incidental and not treatment related.


Uterus weights
The mean gravid uterus weight of the rabbits of test groups 1-3 (6, 20 or 60 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.

The following females were excluded from the above-mentioned calculations:

Test group 0 (0 mg/kg bw/d):
• females Nos. 14, 18, 22, 23 - not pregnant

Test group 1 (6 mg/kg bw/d):
• females Nos. 43, 48 - not pregnant
• female No. 31 - died intercurrently

Test group 2 (20 mg/kg bw/d):
• females Nos. 53, 60, 65, 75 - not pregnant

Test group 3 (60 mg/kg bw/d):
• females Nos. 77, 80, 88 - not pregnant
• female No. 91 - sacrificed after abortion


Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).

Number of abortions:

effects observed, non-treatment-related

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

effects observed, non-treatment-related

Early or late resorptions:

effects observed, non-treatment-related

Dead fetuses:

effects observed, non-treatment-related

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Female rabbits were placed into the study in four cohorts. Each dose group was represented in each cohort. The conception rate was 84% in the control and mid-dose groups (0 and 20 mg/kg bw/d), 88% in the high-dose group (60 mg/kg bw/d) and 92% in the low-dose group (6 mg/kg bw/d). A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study (according to the test guidelines).

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for pre- and post-implantation losses, the number of resorptions and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

In high-dose doe No. 98 (60 mg/kg bw/d) 2 early resorptions were recorded after staining the apparently non-pregnant uterus at C-section (i.e. pregnant by stain), resulting in a 100% post- implantation loss for this animal. This isolated finding did not exert a significant effect on the group mean and is regarded as incidental and not treatment related.

Two dead fetuses were found at C-section of high-dose doe No. 93 which is a rare finding but may occur spontaneously in this rabbit strain. As there was no evidence whatsoever for any potential developmental toxicity of the test item in this study, this is considered to be an incidental finding.

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (6, 20 and 60 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal external malformations
One external malformation was recorded for two fetuses, each, in test groups 0 and 1 (0 and 6 mg/kg bw/d) as listed in the table below. In one control fetus it was associated with an additional visceral malformation. However, this finding was an isolated event in single fetuses, thus, it is considered to be incidental. No statistically significant differences of overall incidences were noted between the groups

Individual fetal external malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 3-02 M umbilical hernia
21-09 F a) umbilical hernia
1 (6 mg/kg bw/d) 30-06 F umbilical hernia
35-02 M umbilical hernia
2 (20 mg/kg bw/d) none
3 (60 mg/kg bw/d) none
a) fetus with additional visceral malformations

Total external malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N(%) 2 (1.1) 2 (1.2) 0.0 0.0
Litter
incidence N(%) 2 (9.5) 2 (9.1) 0.0 0.0
Affected
fetuses/litter Mean% 1.3 1.1 0.0 0.0



Fetal external variations
One external variation (paw hyperflexion) was recorded in two control fetuses, one mid-dose fetus and two high-dose fetuses. This finding can be found in the historical control data at a comparable incidence, thus it is considered to be incidental.

Total external variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 2 (1.1) 0.0 1 (0.5) 2 (1.2)
Litter
incidence N (%) 2 (9.5) 0.0 1 (4.8) 2 (10)
Affected
fetuses/litter Mean% 1.1 0.0 0.4 1.3


Fetal external unclassified observations
One unclassified observation, i.e. placentae necrobiotic, was recorded in one fetus of test group 3 (60 mg/kg bw/d). This finding is considered not to be related to treatment.

Total external unclassified observations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 0.0 0.0 0.0 1 (0.6)
Litter
incidence N (%) 0.0 0.0 0.0 1 (5.0)
Affected
fetuses/litter Mean% 0.0 0.0 0.0 0.6

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in single fetuses of all test groups including the control (0, 6, 20 and 60 mg/kg bw/d), as shown in the table below. Two fetuses had associated soft tissue malformations. All findings were considered to be spontaneous in origin and not treatment-related.

No statistically significant differences between the groups were noted. The overall incidences were well within the historical control range of the test facility.

Individual fetal skeletal malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 19-04 M fused thoracic arch, fused rib
1 (6 mg/kg bw/d) 41-01 M additional vertebral arch and corresponding rib, intercostal rib
41-03 M a) cleft sternum
2 (20 mg/kg bw/d) 73-10 F absent lumbar vertebra
3 (60 mg/kg bw/d) 82-07 M severely malformed vertebral column and/or ribs
87-07 M a) severely malformed vertebral column and/or ribs
a) fetus with additional soft tissue malformations


Total skeletal malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 1 (0.5) 2 (1.2) 1 (0.5) 2 (1.2)
Litter
incidence N (%) 1 (4.8) 1 (4.5) 1 (4.8) 2 (10)
Affected
fetuses/litter Mean% 0.4 4.5 0.5 1.1


Fetal skeletal variations
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.

Total fetal skeletal variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 175 (93) 143 (88) 165 (87) 161 (93)
Litter
incidence N (%) 21 (100) 22 (100) 21 (100) 20 (100)
Affected
fetuses/litter Mean% 93.5 86.4 86.2 92.7


For a better overview, all skeletal variations with statistically significant differences between the control and the treated groups were compiled in the table below. All incidences were expressed on a fetus per litter basis.

Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding Test group 0 Test group 1 Test group 2 Test group 3 HCD
Mean % (range)

Misshapen
sacral vertebra 2.1 2.3 5.3 4.8* 5.0 (1.9 - 8.6)

Incomplete
ossification
of talus;
cartilage present 2.6 2.9 2.3 6.7* 2.3 (0.0 - 4.5)

Unossified talus;
cartilage present 0.5 1.2 3.1 5.8* 1.3(0.0 - 2.6)

HCD = Historical control data
* = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])


The findings ‘incomplete ossification of talus’ and ‘unossified talus’ (with present cartilage, respectively) were statistically significantly increased and outside the historical control range in test group 3 (60 mg/kg bw/d). These findings may represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases.

The increased incidence of ‘misshapen sacral vertebra’ in test group 3 was not related to dose and clearly inside the historical control range. Thus, this finding is considered not to be associated with treatment.


Fetal skeletal unclassified cartilage observations
Some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they were assessed as not treatment-related.

Total unclassified cartilage observations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 20 (11) 12 (7.4) 30 (16) 13 (7.5)
Litter
incidence N (%) 11 (52) 8 (36) 12 (57) 5 (25)
Affected
fetuses/litter Mean% 10.7 6.0 14.3 8.5

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations occurred in the test groups 0, 1 or 3 (0, 6 and 60 mg/kg bw/d), as listed in the table below. Three fetuses in different test groups had additional external or skeletal malformations.

The distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. The total incidence of soft tissue malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

Individual fetal soft tissue malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 15-10 F small thymus
21-09 F a) small spleen
24-05 M, 24-06 F, 24-08 M small spleen
1 (6 mg/kg bw/d) 39-04 F small spleen
41-03 M b) cor triloculare
2 (20 mg/kg bw/d) none
3 (60 mg/kg bw/d) 87-07 M b), 87-08 F small spleen
97-05 F small spleen
a) fetus with additional external malformations
b) fetus with additional skeletal malformations

Total soft tissue malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 5 (2.6) 2 (1.2) 0.0 3 (1.7)
Litter
incidence N (%) 3 (14) 2 (9.1) 0.0 2 (10)
Affected
fetuses/litter Mean% 2.0 2.8 0.0 1.8


Fetal soft tissue variations
The examinations of the soft tissues revealed malpositioned carotid branches and an absent lung lobe (Lobus inferior medialis) in all test groups including the control (0, 6, 20 and 60 mg/kg bw/d). Other variations, such as cystic dilatation of the brain and supernumerary branch from aortic arch (test group 2, respectively), dilated cerebral ventricle (test groups 1 and 2), malpositioned carotid origin (test group 0), dilated aortic arch and narrowed pulmonary trunk (test group 1, respectively) and dilated renal pelvis (test group 3) occurred in individual fetuses of the different test groups.

No statistically significant or toxicologically relevant differences between the groups were noted, and the overall incidences were within the historical control range.

Total soft tissue variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 4 (2.1) 7 (4.3) 7 (3.7) 7 (4.0)
Litter
incidence N (%) 4 (19) 6 (27) 6 (29) 4 (20)
Affected
fetuses/litter Mean% 1.7 5.2 4.3 4.1

Fetal soft tissue unclassified observations


Three unclassified soft tissue observations were recorded. A blood coagulum around urinary bladder was seen in two control, one low-dose, two mid-dose and one high-dose fetuses. This finding can be found in the historical control data at a comparable incidence, therefore, it was neither assessed as treatment-related nor as adverse. Furthermore, a discolored spleen was seen in three fetuses of the same high-dose litter, and an infarct of liver was recorded in one fetus of test group 3. These findings are considered not to be treatment-related.

Total soft tissue unclassified observations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 2 (1.1) 1 (0.6) 2 (1.1) 5 (2.9)
Litter
incidence N (%) 1 (4.8) 1 (4.5) 2 (9.5) 3 (15)
Affected
fetuses/litter Mean% 1.6 0.5 1.0 2.5

Description (incidence and severity):

The mean placental weights in test groups 1, 2 and 3 were not influenced by the test substance and were comparable to the control value.

Details on embryotoxic / teratogenic effects:

Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in all test groups (0, 6, 20 or 60 mg/kg bw/d). The distribution of total malformations about the groups was not related to dose.

Six fetuses had more than one malformation. Male control fetus No. 19-04 had a fused thoracic arch and a fused rib, while female control fetus No. 21-09 showed an umbilical hernia and a small spleen. Furthermore, for male low-dose fetus No. 41-03 (6 mg/kg bw/d) a cor triloculare combined with a cleft sternum was recorded. Male low-dose fetus No. 41-01 had malformations affecting the rib cage, i.e. additional vertebral arch and corresponding rib and an intercostal rib. Male high-dose fetus No. 87-07 (60 mg/kg bw/d) showed a small spleen and a severely malformed vertebral column and/or ribs. Lastly, male high-dose fetus No. 82-07 had also a severely malformed vertebral column and/or ribs. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups.

The findings ‘umbilical hernia’ and ‘small spleen’ which were seen in the multiple malformed fetuses, occurred also in further individual fetuses of test groups 0, 1 and 3. Other malformations, such as small thymus (test group 0) and absent lumbar vertebra (test group 2) were scattered observations in individual fetuses. They all were not dose-related and most of them can be found in the historical control data at comparable frequency. An association of these findings to the treatment is not assumed.

Total fetal malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 7 (3.7) 5 (3.1) 1 (0.5) 4 (2.3)
Litter
incidence N (%) 5 (24) 4 (18) 1 (4.8) 3 (15)
Affected
fetuses/litter Mean% 3.2 6.2 0.5 2.3



A spontaneous origin is assumed for the external variations, soft tissue variations and the broad range of skeletal variations which were noted in fetuses of all test groups including controls.

If all different types of variations are summarized, none of the incidences showed a relation to dosing and can be found in the historical control data at comparable frequency.

The only exception: statistically significantly increased findings ‘incomplete ossification of talus’ and ‘unossified talus’ represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases. As these non or incomplete ossifications were solely found in this single location, and no other signs of a delay of skeletal development were noted, a spontaneous origin of these findings is assumed. In addition, such minor developmental delays are considered not to be adverse events.

Total fetal variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 175 (93) 143 (88) 165 (87) 161 (93)
Litter
incidence N (%) 21 (100) 22 (100) 21 (100) 20 (100)
Affected
fetuses/litter Mean% 93.5 86.4 86.2 92.7


A spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups. The distribution and type of these findings do not suggest any relation to treatment.

Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (60 mg/kg bw/d).

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of Hydroxyethyl acrylate to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused beginning evidence of maternal toxicity at a dose of 60 mg/kg bw/d, such as slightly but consistently reduced food consumption as well as reduced body weight/weight gain during the first two treatment weeks.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is
20 mg/kg bw/d.

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is
60 mg/kg bw/d, the highest dose tested.

The test substance is not teratogenic in rabbits at the tested dose levels.

Executive summary:

In a prenatal developmental toxicity study, Hydroxyethyl acrylate was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28).

Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle.

Clinical examinations revealed no toxicologically relevant difference between the animals receiving 6 or 20 mg/kg bw/d Hydroxyethyl acrylate and the controls.

At the high-dose level of 60 mg/kg bw/d slightly, but consistently lower food consumption as well as lower body weight gain during the first two treatment weeks may represent beginning signs of maternal toxicity.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 60 mg/kg bw/d.

Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and postimplantation losses, the numbers of resorptions and viable fetuses. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Groups of 20-29 bred female rats (17-25 pregnant) were exposed to the compound 6h/day on days 6 through 20 of gestation by inhalation. Control animals were exposed concurrently to filtered room air. The test concentrations of 2-hydroxyethyl acrylate were 1, 5, and 10 ppm (corresponding to approx. 4.8, 24.1, and 48.2 µg/L).

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Hydroxyethyl acrylate
- Analytical purity: 95.8 % (GC)
- Source: Roehm, Germany

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l' Arbresle, France)
- Age at study initiation: Young, nulliparous females
- Weight at study initiation: 200-220 g
- Housing: Single in clear polycarbonate cages with stainless-steel wire lids and hardwood shaving as bedding.
- Diet: Food pellets (UAR Alimentation Villemoisson, France), ad libitum
- Water: Filtered tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12 /12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

EXPOSURE
Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow (6-20 m3/h). The chamber temperature was set at 23 ± 2°C, and the relative humidity at 50 ± 5 %. The exposure system delivered, with an infusion pump, a constant rate of liquid chemical from the top of a heated glass column filled with glass beads. Compressed air heated by a glass heater was introduced at the bottom of the glass column in a countercurrent fashion to the liquid flow. The vapourized compound was introduced into the main air inlet pipe of the exposure chambers.
Concentrations of acrylate ester were monitored continuously with a gas-chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. In addition, exposure levels were determined once during each 6-h exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal. The charcoal samples were then desorbed with dichloromethane. The resulting samples were analyzed by gas chromatography using appropriate internal standards. Since 2-HEA has a rather low vapour pressure (0.07 mm Hg at 20 °C), the presence of liquid particles was evaluated at the highest concentration generated (i.e. 25 ppm). Airborn particles were measured with an Aerodynamic Particle Sizer.
No differences in particle counts were observed between the clean filtered air (control) and the vapour-laden air in the exposure chambers.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Glass/stainless-steel inhalation chambers
- Source and rate of air: Test atmospheres were generated through an additional air-flow  rate passed through the fritted disk of a heated bubbler containing 2-hydroxyethyl acrylate.  The vapourized compound was introduced into the main air inlet pipe of the exposure chamber.
- Air flow rate: 6-20 m3/h

TEST ATMOSPHERE
- Brief description of analytical method used: GC/FID
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical concentrations (mean ± SD):
1.1 ± 0.1 ppm (nominal: 1 ppm)
5.0 ± 0.6 ppm (nominal: 5 ppm)
10.6 ± 1.4 ppm (nominal: 10 ppm)

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2-3
- Length of cohabitation: Overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

day 6-20 of gestation

Frequency of treatment:

6h /day

Duration of test:

until gestation day 21

Dose / conc.:

1 ppm (nominal)

Remarks:

corresponding to approx. 4.8 µg/L

Dose / conc.:

5 ppm (nominal)

Remarks:

corresponding to approx. 24.1 µg/L

Dose / conc.:

10 ppm (nominal)

Remarks:

corresponding to approx. 48.2 µg/L

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Exposure concentrations were based on preliminary studies in which severe maternal toxicity (i.e. weight loss during GD 6-13 and pronounced reduction in weight gain during GD 13-21) was observed at 25 ppm hydroxyethyl acrylate. Maternal mortality also occurred at 25 ppm hydroxyethyl acrylate.

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: On gestation day (GD) 0, 6, 13 and 21.

FOOD CONSUMPTION: YES
Food consumption was measured for the intervals GD 6-13 and 13-21.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The uteri were removed and weighed. The number of implantation sites, resorptions, and dead and live fetuses were recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
The number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide (10 %) to detect very early resorptions.

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were preserved in Bouin's solution and examined for internal soft tissue changes. The other half were fixed in ethanol (70 %), eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

Statistics:

Data were presented as mean ± SD. The number of  implantation sites and live fetuses and the various body weights were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett's test if differences were found. The percentages of non-live  implants and  resorptions and the proportions of fetuses with alterations in each litter were evaluated by using the Kruskal-Wallis test, followed by the Dixon-Massey test where appropriate. Rates of pregnancy, fetal sex ratio, and percentage of litters with malformations or external, visceral, or skeletal variations were analyzed by using Fisher's test. Where applicable, least-squares analysis was carried out. For all statistical tests, the level of significance was set a priori at alpha = 0.05.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No test dams died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weight gain during GD 6-13 and absolute weight gain were significantly reduced at 10 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A slight but statistically significant decrease in maternal food consumption was seen at 10 ppm for the entire exposure period.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on maternal toxic effects:

No test dams died. Maternal body weight gain during GD 6-13 and absolute weight gain were significantly reduced at 10 ppm. A slight but statistically significant decrease in maternal food consumption was seen at 10 ppm for the entire exposure period.

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

ca. 0.024 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

There were no significant changes in the numbers of implantations and live fetuses, incidence of non-live implants and resorptions, or fetal body weights across groups. The only malformation observed was a unilateral microphthalmia at 1 ppm. There were no significant changes in the incidence of external, visceral, or skeletal variations.

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 0.048 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Maternal body weights:

 

Concentration [ppm/6h/d]	Maternal body weight GD 6 [g]	Absolute weight gain [g]
0	262 ± 18	34 ± 15
1	261 ± 16	31 ± 6
5	264 ± 18	29 ± 14
10	263 ± 21	15 ± 14**

 

 

Reproductive parameters:

Conc. [ppm/6h/d]	No. of litters	No. of implantation sites/litter	% of non-live implants/litter	% of resorption sites/litter	No. of live fetuses/litter	Average fetal body weight/litter [g]
0	21	14.71 ± 2.53	10.93 ± 13.99	10.93 ± 13.99	13.05 ± 2.91	5.68 ± 0.32
1	19	15.00 ± 3.27	8.93 ± 22.70	8.93 ± 22.70	14.61 ± 2.79	5.71 ± 0.27
5	22	14.91 ± 2.62	7.63 ± 11.02	7.36 ± 10.21	13.82 ± 2.94	5.69 ± 0.32
10	21	15.33 ± 1.53	6.52 ± 6.73	6.52 ± 6.73	14.33 ± 1.80	5.54 ± 0.25

 

 

Concentration [ppm/6h/d]	0	1	5	10
Mean % of fetuses with:
- any malformations/litter	0	0.40 ± 1.68	0	0
- external variations/litter	0.37 ± 1.68	0.40 ± 1.68	0.28 ± 1.33	0.62 ± 1.97
- visceral variations/litter	3.26 ± 9.12	5.16 ± 9.33	12.21 ± 19.56	9.48 ± 19.22
- skeletal variations/litter	19.37 ± 22.58	18.77 ± 19.53	25.05 ± 25.55	14.76 ± 13.58
- any variations/litter	11.60 ± 12.10	12.49 ± 12.85	18.49 ± 15.00	13.02 ± 12.79

 

* ,** Significant differences from the control (0 ppm) value, p< 0.05, and p< 0.01, respectively.

 

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The requirement of developmental toxicity on rabbits for HEA is filled by reading across to the studies on the structural analogues MA and nBA for which data on rabbits is available.and the hydrolysis products of HEA (AA and EG). This approach is further documented in the attached read-across justification.
- While there is no data for HEA on rabbits, there is data available on rats for HEA, HPA, MA and nBA, which all show a comparable toxicity profile (NOAECs 48.2, 54, 35.8 and 130 µg/L, respectively).
- The hydrolysis products of HEA, AA and EG, do not exhibit developmental toxicity in rabbits (NOAEC 0.673 mg/L, NOAEL 2000 mg/kg bw, respectively).
- In addition to the previously included data for MA (BASF 2009), recently an OECD 414 study on rabbits for nBA was completed (Charles River 2017b) which also shows no developmental effects (NOAEL 400 mg/kg bw).


Please see for more information the read-across justification in Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEC

Remarks:

rabbit

Effect level:

15 ppm

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

rabbit_2009 / Correction for molecular weight not necessary.

Key result

Dose descriptor:

NOAEC

Remarks:

rabbit

Effect level:

0.075 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Result read-across CAS No. 79-10-7

Remarks:

rabbit_1993 / Correction for molecular weight not necessary.

Key result

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOAEC

Remarks:

rabbit

Effect level:

45 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

rabbit_2009 / Correction for molecular weight not necessary.

Key result

Dose descriptor:

NOAEC

Remarks:

rabbit

Effect level:

>= 0.673 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Result read-across CAS No. 79-10-7

Remarks:

rabbit_1993 / Correction for molecular weight not necessary.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20-5-2016 to 25-7-2016 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 Jan 2001

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

GLP compliance:

yes

Specific details on test material used for the study:

- Lot No. F534801GB
- Exp. date: 11 Dec 2016
- Colorless, clear liquid

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hra:(NZW)SPF

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, PA
- Age at study initiation: The animals were approximately 7 months old upon receipt.
- Weight at study initiation: 2922 - 3949 g
- Housing: All rabbits were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel-O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). Nesting material was not required because the females were euthanized prior to the date of expected parturition. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly. Kale (1 leaf at each occasion) was provided to each animal daily for environmental enrichment and to aid in maintaining the animal's gastrointestinal health, beginning upon animal receipt and continuing throughout the duration of the study.
- Diet: The basal diet used in this study, PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322, was a certified feed with appropriate analyses performed by the manufacturer. The basal diet was offered in 25-g increments 3 times per day on the day of arrival and in increased amounts over the next few days, until the animals gradually achieved ad libitum status prior to the dose administration period; basal diet was offered ad libitum thereafter.
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum during the study.
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: 1% carboxymethylcellulose (medium viscosity), 0.014% Kolliphor® EL, and 0.0035% hydrochloric acid in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared daily as single formulations for each dosage level and maintained on wet ice, protected from light. The test substance formulations were stirred continuously on wet ice throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 10, 30 and 80 mg/mL (corresponding to dosage levels of 0, 50, 150 and 400 mg/kg/day)
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the 10, 30, and 80 mg/mL dosing formulations and from the middle stratum of the control group dosing formulations prepared on on the first, approximate middle, and last days of preparation on which all groups were dosed. Analysis was performed using a validated gas chromatography method using flame ionization detection. The analyzed concentrations was 85% to 115% of the target concentration.

Details on mating procedure:

The time-mated rabbits were received on gestation day 2, 3, or 4; a breeding record was provided by the supplier.

Duration of treatment / exposure:

gestation days 7 through 28

Frequency of treatment:

once daily

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

Dosage levels were selected based on the range-finding stud (see any other information on materials and methods)

Maternal examinations:

CAGE SIDE OBSERVATIONS
All rabbits were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from the day of receipt through gestation day 29 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration.

BODY WEIGHT
Individual maternal body weights were recorded on gestation days 0 (by supplier under conditions that were not compliant with GLPs, but in accordance with the supplier’s SOPs), 5, and 7-29 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 7-10, 10-13, 13-21, 21-29, and 7-29.

FOOD CONSUMPTION
Individual food consumption was recorded on gestation days 5-29. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.

POST-MORTEM EXAMINATIONS
The laparohysterectomies and macroscopic examinations were performed blind to treatment group. All rabbits were euthanized on estation day 29 by an intravenous injection of sodium pentobarbital via the marginal ear vein. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were co related with the antemortem observations, and any abnormalities were recorded. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Gravid uterine weight was collected and net body weight (the gestation day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
- Number of corpora lutea: The number of corpora lutea on each ovary was recorded.
- Number of implantations: The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in
10% ammonium sulfide solution for detection of early implantation loss.

Fetal examinations:

- Fetal examinations were performed blind to treatment group.
- External examinations: Each viable fetus was examined externally, individually weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements, degrees of autolysis and gross examinations, if possible, were recorded for late resorptions, and the tissues were discarded.
- Soft tissue examinations: Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development
- Skeletal examinations: Following fixation in alcohol, each fetus was stained with Alizarin Red S8 and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
- Head examinations: Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group.
- Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group.
- Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.

Indices:

Postimplantation Loss/Litter = No. Dead Fetuses, Resorptions (Early/Late)/Group / No. Gravid Females/Group

Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%)/ No. Litters/Group

Where: Postimplantation Loss/Litter (%) = No. Dead Fetuses, Resorptions (Early/Late)/Litter/ No. Implantation Sites/Litter x 100

Summation per Group (%) = Sum of Viable Fetuses Affected/Litter (%)/ No. Litters/Group

Where: Viable Fetuses Affected/Litter (%) = No. Viable Fetuses Affected/Litter/ No. Viable Fetuses/Litter x 100

Historical control data:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related clinical findings were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Findings noted in the test substance-treated groups, including decreased defecation and brown material and/or hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All females in the control, 50, 150, and 400 mg/kg/day groups survived to the scheduled necropsy on gestation day 29.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 400 mg/kg/day group, an absence of a mean body weight gain (0 g) was noted on the first day of dose administration (gestation day 7-8) and resulted in a 78.9% lower mean body weight gain in this group compared to the control group during gestation days 7-10 (57 g, 54 g, and 64 g in the control, 50, and 150 mg/kg/day groups, respectively, compared to 12 g in the 400 mg/kg/day group). Mean body weight gains in the 400 mg/kg/day group were similar to the control group for the remainder of the treatment period (gestation days 10-29). As a result of the lower mean body weight gain at the beginning of the treatment period, a lower mean body weight gain was noted at 400 mg/kg/day compared to the control group when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse.
Mean maternal body weight gains in the 50 and 150 mg/kg/day groups and mean body weights, net body weights, net body weight gains, and gravid uterine weights in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 400 mg/kg/day group, lower mean food consumption was noted during gestation days 7 -10 compared to the control group; mean food consumption in this group was similar to the control group for the remainder of the treatment period (gestation days 10-29). As a result of the lower mean food consumption at the beginning of the treatment period, lower mean food consumption was noted in the 400 mg/kg/day group compared to the control group when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences at 400 mg/kg/day were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse.
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50 and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsy on gestation day 29, no test substance-related internal findings were observed at dosage levels of 50, 150, and 400 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Dead fetuses:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal systemic toxicity & maternal developmental toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related external malformations were noted for fetuses in this study. In the 150 mg/kg/day group, one fetus was noted with omphalocele (a portion of the liver protruded through an opening in the umbilicus, remnants of a membranous sac). The aforementioned malformation noted at 150 mg/kg/day occurred in a single fetus, did not occur in a dose-related manner, and the mean litter proportion was not statistically significantly different from the concurrent control group and was within the test lab historical control data range (version 2016.01); therefore, it was not considered test substance-related. No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related skeletal malformations were noted for fetuses at any dosage level. Vertebral anomaly with or without an associated rib anomaly (extra or fused ribs; extra and/or malpositioned arches; extra, malpositioned, absent, small, fused, and/or misshapen centra) was noted for 2 and 1 fetuses in the 50 and 150 mg/kg/day groups, respectively. The aforementioned malformation at 50 and 150 mg/kg/day occurred infrequently, did not occur in a dose-related manner, and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the Charles River Ashland historical control data range; therefore, it was not considered test substance-related.
- No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related visceral malformations were observed for fetuses at any dosage level. Lobular agenesis of the lungs (right accessory lobe absent) was noted for and 3 fetuses in the 50 and 150 mg/kg/day groups, respectively. Because this finding occurred infrequently, did not occur in a dose-related manner, and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the Charles River Ashland historical control data range, this finding was not considered test substance-related. In the control group, one fetus was noted with an absent left kidney and ureter and one fetus was noted with an absent right thyroid gland.
- No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
- A distended stomach was noted for one fetus in the 50 mg/kg/day group. This finding was not classified as either a malformation or developmental variation, was not considered to be test substance-related because it occurred infrequently and in a manner that was not dose-related.

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) available for morphological evaluation were 219(25), 214(24), 199(25), and 214(24) in the control, 50, 150, and 400 mg/kg/day groups, respectively. Malformations were observed in 2(1), 4(4), 5(4), and 0(0) fetuses (litters) in these same respective dosage groups and were considered spontaneous in origin.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: 99.6%
- Impurities (identity and concentrations): Acetic acid 0.07%, acrylic acid dimer 0.08 %, 4-Methoxy phenol 0.15, all others impurities (approx.) 0.08%
- Lot No.: TF2-66011

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products, Inc. (Denver, PA)
- Age at study initiation: 5.5-6 months old
- Weight at study initiation: 2.8-4.0 kg
- Housing: Single in in stainless steel, wire-mesh cages (61 x 61 x 41 cm)
- Diet (ad libitum): AGWAY® PROLAB® Animal Diet (Agway Inc.) Except during exposures
- Water (ad libitum): Tap water (except during exposures)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-21
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

Females assigned to the study were exposed to acrylic acid vapour or filtered air for 6 hours/day during the period of major organogenesis (gestation day -gd- 6 through 18). Filtered air was bubbled through a glass reservoir containing liquid acrylic acid. For all vapour concentrations, a Dwyer Flowmeter was used to measure airflow prior to passing the air through the acrylic acid. The vapour, was introduced into the exposure chambers through 1 inch glass tubing containing stainless steel wool.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of acrylic acid vapour in each exposure chamber was monitored throughout the 15 days of exposure by sampling with XAD-8 sorbent tubes and subsequent analysis using high performance liquid chromatography (HPLC) analysis. The HPLC system was composed of a Model 981 Lambda Max LC Spectrophotometer, a Programmable Systems 680 Gradient Controller, a 712 WISP, and a Model 501 Solvent Delivery System. A Spectra Physics SP4270 computing Integrator provided a record of the chromatograms, chromatographic analyses, and peak height measurement. The concentration in each exposure chamber atmosphere was determined approximately 3 times during each 6-hour exposure. The control chamber was sampled once daily. The nominal concentration was calculated by dividing the total quantity of acrylic acid delivered to the chamber by the chamber airflow rate.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1

Duration of treatment / exposure:

From day 6 to day 18 of gestation

Frequency of treatment:

6 hours/day

Duration of test:

29 days

Dose / conc.:

25 ppm (nominal)

Remarks:

corresponding to approx. 0.075 mg/L. Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (72.06 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG 2005]

Dose / conc.:

75 ppm (nominal)

Remarks:

corresponding to approx. 0.224 mg/L. Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (72.06 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG 2005]

Dose / conc.:

225 ppm (nominal)

Remarks:

corresponding to approx. 0.673 mg/L. Recalculation based on the equation c(mg/m3) = molar mass (g) / molar volume (L) x c(mL/m3) with molecular weight (72.06 g/mol) and molar volume (24.1 L at 20 °C and 1013 hPa) [DFG 2005]

No. of animals per sex per dose:

16

Control animals:

yes, sham-exposed

Maternal examinations:

CAGE SIDE OBSERVATIONS: not specified

DETAILED CLINICAL OBSERVATIONS: Yes
Prior to the exposure period, animals were observed for clinical signs once daily. Preceding and following each exposure, individual does were observed for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were measured on gd 0, 3, 6, 12, 18, 24, and 29.

FOOD CONSUMPTION: Yes
Food consumption was measured daily throughout the study, beginning on gd 3.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: The gravid uterus, ovaries (including corpora lutea), cervix, vagina, and abdominal and thoracic cavities were examined grossly. The right nasal turbinates were examined. Maternal liver and kidney weights were determined.

Ovaries and uterine content:

Each uterus was removed from the peritoneal cavity, weighed, and dissected longitudinally to expose the contents. All live and dead fetuses and resorption sites (early and late) were recorded. Ovaries were removed from the peritoneal cavity and ovarian corpora lutea of pregnancy were counted. Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for confirmation of pregnancy status.

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

After removal from the uterus, all live fetuses received a single lethal intraperitoneal injection of sodium pentobarbital. All live and dead fetuses were weighed and examined externally for variations and malformations including cleft palate. All live fetuses in each litter were examined for thoracic and abdominal visceral abnormalities. The sex of each fetus was determined during dissection by examination of the reproductive organs. One-half of the live fetuses in each litter were decapitated. The heads were fixed in Bouin's solution for subsequent examination of craniofacial structures. All fetuses (50% intact, 50% decapitated) in each litter were eviscerated, air-dried, processed for skeletal staining with alizarin red S and examined for skeletal malformations and variations. All fetal skeletal preparations were retained.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

The unit of comparison was the pregnant doe or the litter. The data for quantitative continuous variables were intercompared for the 3 exposure groups and the control group by use of Levene's test for equality of variances; analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, pooled t-tests were used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by separate variance t-tests for pairwise comparisons.
Nonparametric data were statistically evaluated using the Kruskal-Wallis test, followed by the Mann-Whitney U test when appropriate. Frequency data were compared using Fisher's Exact Test. With the exception of the data analysis for fetal malformations and variations, all statistical analyses were performed using BMDP Statistical Software (Dixon, 1990). For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In the 225 ppm group, individual clinical signs observed including: perinasal wetness beginning as early as the first day of exposure and ending by the second day after the last exposure; perinasal encrustation (from days 14 through 22); and nasal congestion observed during and subsequent to exposures (through Day 22). Nasal congestion was observed in a single doe from the 75 ppm group on day 12 only. There were no clinical signs observed in does during or subsequent to exposures to 25 ppm acrylic acid vapour.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No animal died in any group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean gestational body weights were equivalent across groups throughout gestation. There were no statistically significant exposure-related reductions in weight gain. However, mean body weight losses were observed in all acrylic acid-exposed groups for Days 6 to 12. Losses in the 25 ppm group were not considered to be biologically significant, since body weight gains were greater than control values (by approximately 60 g) during the preexposure period and the reductions for Days 6 to 12 were not associated with consistent reductions in food consumption for the first half of the exposure period. Reduced body weight gain values in the 75 and 225 ppm groups for Days 6 to 12 were considered to be an exposure-related effect since the reductions were coincident with consistent reductions in food consumption for the first five days of the exposure period. Likewise, increased body weight gains in the 75 and 225 ppm groups for Days 18 to 29 were associated with increases in food consumption during the postexposure period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Exposure-related decreases in food consumption were observed in the 75 and 225 ppm groups during the first 5 days of the exposure period. Throughout the remainder of the exposure period, daily food consumption was consistently reduced in the 225 ppm group and the decreases occasionally reached statistical significance. Occasional reductions in daily food consumption were also observed during the exposure period subsequent to Day 11 (Days 17 to 19) in the 75 ppm group. Average food consumption/day calculated for the entire exposure period was reduced in the 225, but not 75, ppm group. Statistically significant increases in food consumption were observed subsequent to the exposure period in the 225 ppm group for Days 23 to 24 and 28 to 29 and in the 75 ppm group for Day 23 to 24. Mean food consumption values for Days 24 to 27 suggested a trend toward increased food consumption throughout the post exposure period for the 225 ppm group. Mean values for the 75 ppm group suggest a trend toward increased food consumption through Day 26. The statistically significant reduction (of approximately 16%) in food consumption for Day 8 to 9 in the 25 ppm group was not considered to be biologically significant based on slightly greater food consumption values (including increases of up to 14%) in the low concentration group prior to exposures. Occasional increases or decreases in food consumption values for the 25 ppm group subsequent to Day 9 were not considered to be exposure related due to the lack of a dose-response pattern of effects.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no exposure related changes in mean body weight at sacrifice, gravid uterine weight, corrected body weight, or corrected weight change. There were no significant effects of exposure on relative and absolute kidney or liver weights. (Apparently slight increases in absolute and relative liver weights in the 75 and 225 ppm groups were due to single animals in each group which had abnormally large livers).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Upon necropsy on Day 29, pertinent findings included ulcerations in the nasal turbinates of 1 female in the 225 ppm group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no effects of exposure on the number of ovarian corpora lutea, the number of total, viable, or nonviable (early and late resorptions and dead fetuses) implantations/litter. Although percent preimplantation loss was statistically significantly increased in the mid and high concentration groups, the increases were not concentration dependent. Percent live fetuses and sex ratio were equivalent across groups.

Dose descriptor:

NOAEC

Effect level:

0.075 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

There was no evidence of developmental toxicity including teratogenicity at any exposure concentration.
Fetal body weights were unaffected by test substance exposure. There were no increases in the incidences of individual external, visceral or skeletal malformations by category, or of total malformations among all groups. There were no increases in the incidences of individual fetal external, visceral, or skeletal variations, of variations by category, or of total variations among all groups.

Dose descriptor:

NOAEC

Effect level:

>= 0.673 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Maternal food consumption:

Gestation day	Group mean food consumption (g)
	0 ppm	25 ppm	75 ppm	225 ppm
6 – 7	191.42	166.96	147.31^**	101.55^**
7 – 8	193.42	169.23	149.51^**	110.35^**
8 – 9	195.13	163.75^*	150.69^**	121.66^**
9 – 10	185.38	166.73	153.59^*	132.49^**
10 – 11	184.98	200.79	154.79^*	134.49^**
12 – 13	180.85	123.21^*	203.10	136.18^**
16 – 17	187.39	186.42	161.23	152.77^*
18 – 19	208.56	178.61	167.23^*	147.33^**
23 - 24	158.19	176.43	204.25^**	199.75^*
28 - 29	134.35	167.32^*	160.41	188.19^**

^* Significantly different from control group (p<0.05.)

^**Significantly different from control group (p<0.01.)

Maternal body weights and weight changes:

Gestation day	Gestational mean body weight changes (g)
	0 ppm	25 ppm	75 ppm	225 ppm
0 - 3	-34.08 (150.76)	-7.47 (152.69)	-6.83 (150.42)	-43.38 (214.46)
3 - 6	203.77 (96.19)	240.45 (76.84)	206.55 (96.92)	163.45 (251.41)
6 - 12	68.43 (65.47)	-18.87 (93.75)	-37.67 (93.47)	-41.06 (201.25)
12 - 18	146.67 (69.62)	160.54 (72.07)	123.51 (63.56)	148.45 (65.59)
18 - 24	112.36 (73.58)	150.13 (109.11)	178.41 (73.84)	176.15 (86.22)
24 - 29	26.65 (89.84)	51.55 (125.51)	64.32 (87.17)	143.49^**(96.91)

^**Significantly different from control group (p<0.01.)

Numbers in parentheses indicate standard deviation

Gestational parametres:

Concentration (ppm)	0	25	75	225
Corpora lutea (mean)	8.4	9.1	9.5	8.8
Implantations (mean)	8.6	8.8	8.2	8.5
Viable implants (mean)	8.4	8.6	7.5	7.9
Percent live fetuses (mean)	97.8	98.4	91.3	95.0
Dead implantations (mean)	0.1	0.1	0.0	0.1
Mean weight of live fetuses (g)	43.99	42.44	42.44	42.93

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Proposal for updating Guideline 414: Prenatal Developmental Toxicity Study (22 Jan 2001)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

other: Corrigendum to EC Commission Directive 2004/73/EC, Part B: Methods for the determination of toxicity: Prenatal Developmental Toxicity Study; Official Journal of the European Union, No. L 216, pp. 227-235 (29 Apr 2004)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test substance: Methyl Acrylate (MA)
- CAS No.: 96-33-3
- Purity: 99.9 area-%
- Physical state/Appearance: Liquid /colorless, clear
- Homogeneity: Given
- Batch No.: 010653eda0 Verkaufstank 02 vom 20.11.2007
- Storage conditions: Refrigerator (KS), avoid temperatures >35 °C, under light exclusion, light sensitive
- Stability: Expiry date: 27 Oct 2008

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 16-24 weeks
- Weight at study initiation (pregnant animals): 2202-2829 g
- Housing: Singly in type 12.2395.C stainless steel wire mesh cages. During exposure, the animals were kept singly in special mesh cages (40x13x16cm) from BASF SE.
- Diet: Pelleted “Kliba maintenance diet for rabbit & guinea pig, GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum. During exposure, no food was supplied.
- Water: Tap water ad libitum. During exposure, no drinking water was supplied.
- Acclimation period: approx. 7-14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Remarks:

conditioned supply air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass steel inhalation chamber, volume of 1.4 m³ (BASF SE)
- Method of holding animals in test chamber: animals were kept singly in wire cages located in the inhalation chamber
- System of generating inhalation atmospheres: Generator systems: two-component atomizers (BASF SE); continuous infusion pumps PERFUSOR (B. Braun); Glass mixing stages (BASF SE); glass vaporizers with thermostat (BASF SE). Generation procedure: The test substance was used unchanged. For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer at a constant rate by means of the piston metering pump. The vapor was generated by spraying the substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system.

TEST ATMOSPHERE
- Brief description of analytical method used: GC analysis of absorption samples
- Samples taken from breathing zone: yes, immediately adjacent to the animals' noses

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Calculation of nominal concentrations: The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.
Online monitoring of the atmospheric concentration: The constancy of concentrations in the inhalation atmospheres were monitored continuously by calibrated online total hydrocarbon analyzer (TESTA FID 2010T in test group 0 and TESTA FID 123 in test groups 1-3) in all test groups including control. Using line recorders the measurements were recorded (see examples in Figure 003 in the APPENDIX) and the signals were transferred to the automated measuring system. The long-term drift of the online devices were checked by a weekly measurement of certificated test gas. To control the correctness of the calibrated devices and the identity of the test substance in the atmosphere, weekly two absorption samples per concentration were drawn from the atmospheres and were analyzed by gas chromatography (GC). The measured values of test group 0 served as blind control. The blind values were mainly the animals' gas emission and, in minor amount, directly from the atmosphere. Therefore, the concurrent blind values were substracted from the measured values. From the corrected daily mean values of each concentration, mean concentrations and standard deviations for the entire study were calculated.

Details on mating procedure:

- Impregnation procedure: artificial insemination
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:

6 hours on workdays over a time period of 23 consecutive days (gestation days (GD) 6–28)

Frequency of treatment:

daily on workdays

Duration of test:

Until GD 29

Dose / conc.:

4.9 ppm (analytical)

Remarks:

equivalent to 17.4 mg/m³ (analytical); 21.9 mg/m³ (nominal)

Dose / conc.:

15.7 ppm (analytical)

Remarks:

equivalent to 55.3 mg/m³ (analytical); 57.2 mg/m³ (nominal)

Dose / conc.:

44.2 ppm (analytical)

Remarks:

equivalent to 155.6 mg/m³ (analytical); 176.5 mg/m³ (nominal)

Dose / conc.:

5 ppm

Remarks:

target concentration

Dose / conc.:

15 ppm

Remarks:

target concentration

Dose / conc.:

45 ppm

Remarks:

target concentration

No. of animals per sex per dose:

25 inseminated female Himalayan rabbits

Control animals:

yes, sham-exposed

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0 29). During pre-flow period and on the day of necropsy the animals were examined for clinical symptoms at least once a day. During the exposure period, a clinical inspection of each animal was performed at least three times a day (before, during and after exposure). During the exposure procedure a groupwise examination was conducted.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated.
- Corrected (net) body weight gain: The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
- The food consumption was determined daily on GD 1–29. Because of the malfunction of a balance, the food consumption was not properly recorded on 24 June 2008and the values from this day were not used for the calculation of means and were specified as “not measured / no value determinable”.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: weight assessment: lungs; histopathology: nasal cavities, larynx, trachea, lungs, mediastinal lymph nodes, all gross lesions

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as: 1) live fetuses; 2) dead implantations: a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non pregnant animals and the empty uterus horn in the case of single horn pregnancy); b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible); c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
- Head examinations: Yes, half per litter

Statistics:

DUNNETT-test (two-sided), FISHER'S EXACT test (one-sided), WILCOXON-test (one-sided), KRUSKAL-WALLIS test (two-sided)

Indices:

Calculations of conception rate and pre- and postimplantation losses were carried out:
The conception rate (in %) was calculated according to the following formula: (number of pregnant animals)/(number of fertilized animals) x 100
The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula: (number of corpora lutea – number of implantations)/(number of corpora lutea) x 100
The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula: (number of implantations – number of live fetuses)/(number of implantations) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical signs or any disturbances of the general behavior were observed in any rabbit during the entire study period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- The average body weights and body weight gain were comparable among control and treated groups (5, 15 and 45 ppm) during the entire study. All observable differences in the treated groups in comparison to the controls are without any biological relevance. This statement includes the statistically significantly decreased body weight gain in test group 3 on GD 9-11.
- The results of the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) revealed no difference of biological relevance between the test substance-treated groups and the control group. Mean carcass weights remained also unaffected by the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The average food consumption was comparable to the control group in all test groups (5, 15 and 45 ppm) and did not show a test substance-related impairment. Differences between control rabbits and test substance-treated rabbits did not show a relation to dosing and were considered to be without biological relevance. This overall statement is true in spite of the slightly but significantly lower high-dose value on GD 23-24.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of the animals of all test groups (5, 15 and 45 ppm) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance. Considering the fluctuations in the mean number of live fetuses/doe, they reflect the normal degree of variation for rabbits of the strain used in this study.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The conception rate reached 96% in test groups 0, 2 and 3 (0, 15 or 45 ppm) and 100% in test group 1 (5 ppm). Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 24-25 pregnant rabbits per group had implantation sites in the uterus. There were no test substance related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the post implantation losses, the number of resorptions and viable fetuses.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Gestational parameters in the various test groups were within the normal range for animals of this strain and age

Details on maternal toxic effects:

Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, corrected (net) body weight gain and summary of reproduction data. On female each of test group 0 (0 ppm), test group 2 (15 ppm), and test group 3 (45 ppm)were excluded from the above-mentioned calculations since they were not pregnant. Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).

Dose descriptor:

NOAEC

Effect level:

15 ppm

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean placental weights in test groups 1, 2 and 3 (5, 15 and 45 ppm) were comparable to the controls.
The mean fetal weights of all treated groups were not influenced by the test substance. Neither female nor male fetal weights showed statistically significant or biologically relevant differences between the test substance-treated groups and the controls.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (5, 15 and 45 ppm) was comparable to the control fetuses. Observable differences were without biological relevance.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Fetal external malformations: Three external malformations, which were associated with corresponding soft tissue and skeletal malformations, were recorded for single fetuses of test groups 1 and 2 (5 or 15 ppm). Because a dose-response relationship was missing these findings were considered to be spontaneous in nature.
- Fetal external variations: One external variation (paw hyperflexion) occurred in single fetuses of all treated groups and the control. The incidences did not demonstrate a dose-response relationship and were comparable to the historical control data. Thus an association of this finding to the treatment is not assumed.
- Fetal external unclassified observations: One unclassified external observation, i.e. blood coagulum around placenta, was recorded for one fetus of the control group and was spontaneous in nature.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Fetal skeletal malformations: Malformations of the fetal skeletons were observed in fetuses of all test groups including the controls (0, 5, 15 and 45 ppm). Neither statistically significant differences between treated groups and control nor a dose-response relationship were noted. When calculated on a fetus per litter basis, the overall incidence of skeletal malformations was comparable to the historical control data. This is also true for the severely fused sternebrae (bony plate), where the incidence of affected fetuses per litter in the high-dose group was also slightly but statistically significantly higher than the concurrent control.
- Fetal skeletal variations: For all test groups, variations were detected in different skeletal structures with or without effects on corresponding cartilaginous structures. The observed skeletal variations were related to various parts of the fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data. Two isolated skeletal variations were statistically significantly higher than the concurrent and/or outside the historical control (on a fetus per litter basis). These findings are delays of ossification which is reversible and does not affect the morphology of the cervical vertebra as it becomes obvious by the unchanged underlying cartilage. Such slight retardations of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain. Also, the increased incidences are not at all related to the dose. Thus, these findings are regarded to be of no biological relevance.
- Fetal skeletal unclassified cartilage observations: Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing and were considered to be spontaneous in nature.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Fetal soft tissue malformations: The examination of the soft tissues revealed three malformations in single fetuses of all treated groups (5, 15 and 45 ppm). Although no findings were observed in the control and the rate of affected fetuses per litter was significantly higher in the low- and mid-dose groups, no dose related increase of the incidence of soft tissue malformations was noted. Apart from the single incidental case of small cerebrum the findings occurred at incidences comparable to the historical control data. Thus an association of the soft tissue findings to the treatment is not assumed.
- Fetal soft tissue variations: Three soft tissue variations, such as absent lung lobe (lobus inferior medialis), malpositioned carotid branch and dilated cerebral ventricle, were detected in each test group including the controls without relation to dosing. Neither statistically significant differences between the test groups nor differences to the historical control data were noted.
- Fetal soft tissue unclassified observations: Unclassified soft tissue observations, such as discolored kidney, blood coagulum around urinary bladder and hemorrhagic ovary, were recorded for some fetuses of all test groups (0, 5, 15 and 45 ppm). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance-induced effect is not assumed

Details on embryotoxic / teratogenic effects:

Summary of all classified fetal external, soft tissue and skeletal observations: Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. They did neither show a consistent pattern since a number of morphological structures of different ontogenic origin were affected nor a clear dose-response relationship. Furthermore, the overall incidences were comparable to the historical control data. Thus, non of the malformations was considered to be related to the treatment. One external (paw hyperflexion), three soft tissue (absent lobus inferior medialis, malpositioned carotid branch and dilated cerebral ventricle) and a broad range of skeletal variations occurred in all test groups including the controls. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter did not show a relation to dosing. In addition, they can be found at a comparable frequency in the historical control data. Therefore, they were not considered to be related to the treatment.

Dose descriptor:

NOAEC

Remarks:

developmental toxicity

Effect level:

45 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

- Study means and standard deviations of test substance concentrations:

Test group	Target concentration		Measured concentration (FID)				Nominal concentration (mg/m³)	Effectiveness of vapor generation (%)
			(ppm)		(mg/m³)
	ppm	mg/m³	Mean	SD	Mean	SD
0	0	0	-	-	-	-	-	-
1	5	17.6	4.9	1.1	17.4	3.9	21.9	79.5
2	15	52.8	15.7	1.8	55.3	6.2	57.2	96.8
3	45	158.4	44.2	1.2	155.6	4.3	176.5	88.2

- = not measured

- Occurrence of statistically significantly increased fetal skeletal variation (expressed as mean percentage of affected fetuses/litter):

Finding	Test group 0 0 ppm	Test group 1 5 ppm	Test group 2 15 ppm	Test group 3 45 ppm	HCD Mean % (range)
Incomplete ossification of cervical centrum; unchanged cartilage	1.9	8.9**	4.9	6.1	2.2 (0.0–5.0)
Incomplete ossification of sacral arch; cartilage present	0.0	0.7	1.9*	1.2	0.3 (0.0–2.2)

ppm = parts per million; HCD = Historical control data

* = p≤0.05, ** = p≤0.01 (Wilcoxon-Test [one-sided])

- Total fetal malformations: 

		Test group 0 0 ppm	Test group 1 5 ppm	Test group 2 15 ppm	Test group 3 45 ppm
Litter Fetuses	N N	24 144	25 171	24 150	24 136
Fetal incidence	N (%)	3 (2.1%)	8 (4.7%)	7 (4.7%)	6 (4.4%)
Litter incidence	N (%)	3 (13%)	6 (24%)	6 (25%)	6 (25%)
Affected fetuses/ litter	Mean%	1.9	4.0	4.6	3.7

ppm = parts per million; N = number; % = per cent

- Total fetal variations:

		Test group 0 0 ppm	Test group 1 5 ppm	Test group 2 15 ppm	Test group 3 45 ppm
Litter Fetuses	N N	24 144	25 171	24 150	24 136
Fetal incidence	N (%)	96 (67%)	123 (72%)	119 (79%)	83 (61%)
Litter incidence	N (%)	24 (100%)	25 (100%)	24 (100%)	23 (96%)
Affected fetuses/litter	Mean%	68.8	71.9	77.9	60.5

ppm = parts per million; N = number; % = per cent

Conclusions:

The administration of Methyl Acrylate (MA) to pregnant Himalayan rabbits via inhalation had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm). The NOAEL for maternal toxicity is 15 ppm. The NOAEL for prenatal developmental Toxicity is 45 ppm. No adverse fetal findings of toxicological relevance were evident at any dose.

Executive summary:

The study was performed according to the proposal for updating OECD Guideline 414: Prenatal Developmental Toxicity Study (22 Jan 2001) in compliance with GLP.

Methyl Acrylate (MA) was administered to pregnant Himalayan rabbits via inhalation from implantation to one day prior to the expected day of parturition (GD 6-28).

There were no test substance-related effects on the does concerning food consumption, gross/net body weight, gestational parameters, uterine, placental and lung weights, as well as necropsy observations up to and including a dose of 45 ppm. The test substance caused a severe degeneration and atrophy of the olfactory epithelium at at least one focal area in the nasal cavity (distal levels III and/or IV) at the high-dose level (45 ppm). Though being local effects such massive findings in the respiratory tract are likely to cause a considerable amount of distress in the affected maternal animals. Since distress is supposed to influence maternal homeostasis this is considered to be a significant adverse effect on maternal organism.

Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Methyl Acrylate (MA) had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm).

Conclusion: The NOAEL for maternal toxicity is 15 ppm. The NOAEL for prenatal developmental Toxicity is 45 ppm. No adverse fetal findings of toxicological relevance were evident at any dose.

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Assessing the potential of EG, when administered by gavage during major organogenesis, to cause maternal and developmental toxicity in rabbits, a common non-rodent test animal species.

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight: The range of actual body weights for females on GD 0 was 2470 to 4460 g
- Housing: Inseminated females were individually housed in stainless steel cages with mesh flooring
- Diet: Certified rabbit chow, ad libitum
- Water: filtered water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 °C
- Humidity (%): 54.1% (range of 26.8 to 81.9%)
- Photoperiod (hrs dark / hrs light): Animal room lights were on from 7 to 19 hr for females and from 7 to 21 hr for males

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Artificially inseminated New Zealand white does were dosed daily with EG in vehicle or with the deionized/distilled water vehicle on the mornings of GD 6-19. Treatment was by gavage using a stainless steel dosing tube. A dose volume of 5 mL/kg body wt was used for all groups. The volume administered was adjusted according to daily body weights. All formulations were 93 -107% of theoretical for predosing analyses/for the postdosing analyses. All formulations were 91.6-109% of the predosing measured concentrations.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

To induce ovulation, females received an intravenous injection of Pregnyl (0. 1 ml/kg) immediately prior to insemination. The day of insemination was designated as Gestational Day (GD) 0. Females were assigned to dose groups (23 -24 per group) by stratified randomization on GD 0 so that body weights did not differ among groups within any individual replicate. The study was performed in two replicates (11-12 inseminated females per dose per replicate) with two consecutive breeding days in each replicate. The last breeding date for the first replicate and the first breeding date for the second replicate were 5 weeks apart.

Duration of treatment / exposure:

gestation day 6 to19

Frequency of treatment:

daily

Duration of test:

until gestation day 30

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

23 - 24

Control animals:

yes, concurrent no treatment

Details on study design:

The doses chosen were based on preliminary studies with nonpregnant rabbits and information from rodents which indicated developmental effects at much lower doses than maternal effects. Analyses of the dosing formulations indicated that they were homogeneous and stable for at least 3 weeks.

Maternal examinations:

Inseminated females were weighed on GD 0, 6-19, 25, and 30. Females were observed and maternal water consumption was measured daily throughout gestation GD 0-30. All surviving inseminated females were killed at scheduled necropsy on GD 30 by iv injection of a euthanasia solution into the marginal ear vein. The maternal liver, kidneys and intact uterus were weighed and corpora lutea counted. Uteri which presented no visible implantation sites were stained with ammonium sulfide (10%) to detect very early resorptions. Maternal kidneys were bisected and fixed in 10% neutral buffered formalin. Kidneys from 10 randomly selected does from the control through the 1000 mg/kg/day EG groups and all the kidneys from the 2000 mg/kg/day EG group as well as kidneys from all does which died during study were sectioned, stained with hematoxylin/eosin and evaluated histologically. The sections were also examined under polarized light for oxalate crystals.

Fetal examinations:

Live fetuses were dissected from the uterus and euthanized. They were weighed, examined for external morphological abnormalities including cleft palate and dissected for visceral examination and determination of sex by a fresh tissue dissection technique. Half of the fetuses were decapitated after dissection: the heads were fixed in Bouin's solution and then examined by a freehand sectioning technique. All fetal carcasses were skinned, cleared, stained with Alcian blue/alizarin red S. and examined for skeletal malformations and variations.

Statistics:

Analyses were performed using the doe or the litter as the experimental unit. General Linear Trend Models procedures were applied for the analysis of variance (ANOVA) of maternal and fetal parameters. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data and Bartlett's test for homogeneity of variance was performed on all data to be analysed by ANOVA.

Clinical signs:

no effects observed

Description (incidence and severity):

No clear treatment-related clinical signs of toxicity were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Maternal toxicity was observed at 2000 mg/kg/day, expressed as 42.1% mortality (8 of 17 pregnant animals). The 8 does which died at 2000 mg/kg/day died on GD 9 (one doe) and 11 (two does) and on GD 13, 14, 19, 21 and 25 (one doe each).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Periodic maternal body weights and weight changes were statistically equivalent across all groups for all intervals evaluated.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Maternal water consumption, expressed as g/animal/day or as g/kg/day was also statistically equivalent across all groups for all intervals, although water consumption appeared slightly increased for all EG-dosed groups during the treatment period but not in a manner related to dose.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

At scheduled necropsy on GD 30 there were no significant effects of treatment on corrected (for uterine weight) maternal gestational weight change, gravid uterine weight, liver weight, or kidney weight at any dose. However, maternal absolute kidney weight (but not relative weight) was slightly increased at 2000 mg/kg/day to 106.3% of the control value for the right kidney (left kidney value was 107.6% of control).

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histologic evaluation of maternal kidneys revealed treatment-related renal lesions only at 2000 mg/kg/day. The lesions were limited to the cortical renal tubules and included intraluminal crystals (appearance consistent with oxalate), epithelial necrosis, and tubular dilatation and degeneration. The most severe findings, crystals (designated "marked") and necrosis, were observed in the does which died on study, but the renal tubular necrosis observed in these animals was not a postmortem event. The cause of death in these animals was determined to be renal failure.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

An increase in early deliveries with three does at 2000 mg/kg bw (and one each at all other doses) was observed. One dose at 2000 mg/kg/day aborted on GD 20 (no litters were aborted at any other doses). lncreases in early deliveries and abortion are usually considered indicative of maternal stress in rabbits.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on any gestational parameters, including no effects on number of ovarian corpora lutea, on total non-live or live implantation sites per litter, or on pre- or post-implantation loss. There was a statistically significant increase in the number of corpora lutea at 500 mg/kg/day associated, as expected, with a slight increase in the number of implantation sites/litter and a slight increase in live litter size. This finding was not observed at higher doses and is considered most likely due to biologic variation. The values at 500 mg/kg/day are still well within historical control values for these parameters.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Pregnancy rate was high and equivalent across all groups from 0 - 1000 mg/kg/day (95.5, 95.7, 91.3, and 95.2%, respectively); pregnancy rate was slightly lower (8 1.8%) at 2000 mg/kg/day.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

not examined

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no statistically significant or biologically relevant differences among groups for prenatal mortality, expressed as resorptions or dead fetuses, or for prenatal toxicity, expressed as fetal body weight/litter for all fetuses or for male and female fetuses separately.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no treatment-related alterations in incidence of malformations pooled as external, visceral (including craniofacial), or skeletal, or as total malformations or variations. Examination of fetal malformations and variations by individual findings also indicated no findings which were treatment- or dose-related and none which appeared predominantly or exclusively at higher doses.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The sensitivity of NZW rabbits relative to that of Sprague-Dawley rats and Swiss mice for maternal and developmental toxicity from gavage administration of EG during organogenesis can be determined for maternal toxicity: rabbits > mice > rats, and for developmental toxicity: mice > rats > rabbits.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity:

In a prenatal developmental toxicity study (BASF 2021), HEA was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28). Clinical examinations revealed no toxicologically relevant difference between the animals receiving 6 or 20 mg/kg bw/d Hydroxyethyl acrylate and the controls. At the high-dose level of 60 mg/kg bw/d slightly, but consistently lower food consumption as well as lower body weight gain during the first two treatment weeks may represent beginning signs of maternal toxicity. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 60 mg/kg bw/d. Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and postimplantation losses, the numbers of resorptions and viable fetuses. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age. Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development.

 

In another study where the developmental toxicity of seven acrylates was investigated (Saillenfait 1999), groups of 25 pregnant rats were exposed to 0, 1, 5 or 10 ppm HEA vapor (corresponding to approx. 0.0048, 0.0241, and 0.0482 mg/L) for 6 hrs/day from days 6 through 20 of gestation. Maternal toxicity was demonstrated at 10 ppm as a statistically significant decrease in maternal body weight gain over the entire exposure period, which was also statistically different from controls on days 6-13. A statistically significant decrease in food consumption as compared to controls was also observed for the 10 ppm group on days 6-21.Uteri were removed and weighed, and the number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide to detect very early resorptions. Live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were preserved in Bouin’s solution and examined for internal soft tissue changes. The other half were fixed in ethanol, eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination.There were no treatment-related increases in the number of implants and the number of live fetuses, in the incidence of non-live implants and resorptions, or fetal body weights across groups. The only malformation observed was a unilateral microphthalmia at 1 ppm. There were no significant changes in the incidence of external, visceral, or skeletal variations.

In the study of Saillenfait (1999) additionally the source substance HPA, MA and nBA were investigated. HPA was exposed at dose levels of 0 (air), 5.4, 27 and 54 µg/L for 6 h/day,

MA was exposed at dose levels of 0 (air), 8.9, 17.9 and 35.8 µg/L for 6 h/day and nBA at0.52; 1.05; 1.57 mg/L for 6 h/day. No developmental toxicity was observed in these studies up to the highest dose tested.

No developmental toxic effect was observed in a study according OECD 422, HEA was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 12, 40 and 120 mg/kg body weight/day (mg/kg bw/day) to screen for potential systemic, reproductive and developmental toxicity (BASF SE, 2020).

 

n-Butyl acrylate: For nBA additional studies have been performed in rodents. Sprague Dawley rats were exposed to nBA vapor concentrations of 25, 135 and 250 ppm (corresponding to approx. 0.13; 0.71; 1.31 mg/L) for 6 hours per day on days 6 to 15 of gestations (BASF 1979d). Inhalation of 135 and 250 ppm caused a significant reduction in maternal body weight gain, as well as irritation to the nose and eyes due to the known irritative nature of this test substance to mucous membranes. Following the exposure until termination the animals recovered and the weight gain was comparable to the controls. The two highest concentrations caused a dose-dependent increase in post-implantation loss (i.e. increase in resorptions). 25 ppm did not lead to any signs of maternal toxicity and had no effect on the implants. No substance-related morphological alterations, especially no malformations were observed in the fetuses at any concentration. The NOAEC for maternal and developmental toxicity was 25 ppm (0.13 mg/L), and the NOAEC for teratogenicity was 250 ppm (1.31 mg/L), the highest concentration examined. It is noteworthy to mention that no histopathological examination of the respiratory tract of the dams was performed, as it was not required by the guideline. However, with regards to the observed respiratory irritation in the subchronic inhalation study at comparable concentrations, the occurrence of clear effects on the respiratory tract as evidence for maternal toxicity can be considered as very likely, especialelopmental toxicity (BASF SE, 2020). A detail description is given in the Section Fertility abovely as irritation was already noted clinically.

 

In a prenatal developmental toxicity study with low reliability due to excessive dose levels by far exceeding the maximum dose recommended by the respective OECD guidelines and doses causing maternal lethality, pregnant CD-1 mice received nBA dissolved in cottonseed oil at dose levels of 0, 100, 1000, 1500, 2000, 2500, 3000 and 4000 mg/kg bw orally by gavage from gestation day 6 to day 15. No animal survived at the high dose. At 3000 and 2500 mg/kg bw 2/30 animals died; at 2000 mg/kg bw 1/29 died; at 1500 mg/kg bw 1/27 died; and at 1000 mg/kg bw 1/30 died. At 1500 mg/kg bw and above, mean maternal body weight gain and concurrently, mean fatal body weights were significantly reduced. Most likely due to the maternal stress, the percentage of resorptions was significantly increased at 2500 and 3000 mg/kg bw. In the control group as well as in at 100, 1000, 1500 and 2000 mg/kg bw, variations and malformations occurred sporadically at different sides (i.e. single cases of cleft palate, fused ribs, fused sternebrae, fused arches, extra arches, branched ribs) in a non-dose-dependent manner. At maternally lethal dose levels of 2500 mg/kg bw and 3000 mg/kg bw the number of fetuses with external and skeletal malformations and variations (cleft palate, exencephaly, open eyes, fused arches, fused ribs) was significantly increased. The NOAEL for maternal toxicity was 100 mg/kg bw. The NOAEL for developmental toxicity was 1000 mg/kg bw and the NOAEL for teratogenicity was 2000 mg/kg bw. It is noteworthy to mention that currently the internationally accepted limit dose level is 1000 mg/kg bw. Thus, the NOAEL for developmental toxicity was at this limit dose level, while the NOAEL for teratogenicity was 2-fold above (Rohm & Haas 1979).

 

Acrylic Acid:

For the common metabolite AA an OECD 414 study is available. Groups of 30 pregnant Sprague-Dawley rats were exposed (6 h/d, whole-body) to atmospheres containing AA at 0, 40, 120, and 360 ppm (corresponding to approx. 0, 0.12, 0.36 and 1.08 mg/L) during days 6 to 15 of gestation in a developmental study according to OECD TG 414. After exposure the dams were observed up to day 20 of gestation (BASAF 1983 The animals ’body weight and food consumption were determined on gestation day 0 and subsequently on every third day up to gestation day 20. After sacrifice dams were subjected to a gross pathological examination. After external examination of each foetus their body weights and lengths were measured and they were further processed for skeletal and visceral examination.In the dams, irritation of the respiratory tract and the eyes was observed in the highest dose group. A dose-related reduction in food and water intake resulting in a decrease in body weight gain was observed in the 120 and 360 ppm groups. Also in the 40 ppm group a slight but statistically significant effect was seen on body weight gain (between day 0 and 20 minus uterus weight) of the dams (10 % reduction as compared to the control). Since this finding at 40 ppm was the only effect observed at this dose level and with unclear biological relevance, it was concluded that the NOAEC for maternal toxicity was 40 ppm (= approx. 0.12 mg/L).No effects on reproductive performances were observed. There were no signs of group-related trends or significant differences between groups in terms of pre-implantation losses, live foetuses, or resorptions. There were also no signs of group-related differences in the incidences of abnormalities, variations, or retardations in the foetuses in terms of general appearance, foetal body weights and the conditions of the internal organs or the skeleton. Thus, the NOAEC for developmental toxicity in rats was set at 360 ppm = approx. 1.080 mg/L.

 

Ethane-1,2-diol:

 

For the HEA specific metabolite ethane-1,2-diol, multiple prenatal developmental studies have been performed in rat and mice. The substance caused developmental toxicity characterized by craniofacial and axial-skeletal malformations and variations in mice and rats when administered by oral gavage during the period of organogenesisTyl et al. (1985, 1988, 1995a,b), Neeper-Bradley et al (1990 and 1995), NTP (1988), Price et al. (1984, 1985), Maronpot et al. (1993).

Subsequent investigations, bothin vivoandin vitro, have established that the developmental toxicity ofethane-1,2-diol in rats is related to the accumulation of glycolic acid (GA) in the blood and metabolic acidosis. The toxicity of GA, both in vivo and in vitro, is exacerbated under acidic conditions and is related to fetal disposition. Whenethane-1,2-diolwas administered to rats and rabbits at a developmentally toxic dose (1000 mg/kg bw/d, see section 5.4.3), GA was found to be preferentially distributed into the rat embryo compared to the maternal blood (embryo:blood concentration 1.54) whereas this was not the case in the rabbit (embryo:blood concentration 0.31). Recent investigation demonstrated that GA uptake into the rat embryo occurs predominantly by a specific, pH-dependent, active uptake transporter protein, consistent with the proton-linked monocarboxylate transporters (MCT). Given that GA disposition between the maternal blood and the embryo is driven by the polarity of MCT1 and MCT4 isoforms in the placenta, that GA is sequestered in the rat and not the rabbit, and that the rabbit and human placenta show similar polarity to each other and opposite to that of the rodent, it is proposed that the rabbit is the most appropriate species for the basis of classification. As such, and since EG is not a developmental toxicant in the rabbit, no classification is warranted.

 

Methyl acrylate:

For MA an OECD414 study was performed in rabbit as second species(BASF 2009). 25 inseminated female Himalayan rabbits per group were whole-body exposed for 6 hrs/day, 5 days/week over a time period of 23 consecutive days (gestation days (GD) 6–28) to MA vapors at target concentrations of 0, 5, 15, and 45 ppm. Analytical concentrations of 4.9, 15.7, 44.2 ppm (corresponding to approx. 0.0174, 0.0553, 0.1556 mg/L) were measured. On gestation day 29 the does were sacrificed and submitted to gross and histopathological examination (nasal cavities, larynx, trachea, lungs, mediastinal lymph nodes, all gross lesions). Examinations of ovaries and uterine content of the does included: determination of the weight of the unopened uterus, of the number of corpora lutea, of the number and distribution of implantation sites, and calculations of conception rate and pre- and post-implantation losses. Fetal examinations were performed on all fetuses per litter (external, soft tissue, skeletal) except head examinations that were done on half of the fetuses per litter.There were no test substance-related effects on the does concerning food consumption, gross/net body weight, gestational parameters, uterine, placental and lung weights, as well as necropsy observations up to and including a dose of 45 ppm. The test substance caused a severe degeneration and atrophy of the olfactory epithelium at at least one focal area in the nasal cavity (distal levels III and/or IV) at the high-dose level (45 ppm). Though being local effects, such massive findings in the respiratory tract are likely to cause a considerable amount of distress in the affected maternal animals. Since distress is supposed to influence maternal homeostasis, this is considered to be a significant adverse effect on the maternal organism. The NOAEC for maternal toxicity was 15 ppm (0.0553 mg/L).Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. MA had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm). Thus, the NOAEC for developmental effects (fetotoxicity) and the NOAEC for developmental effects (teratogenicity) was the highest concentration tested of 45 ppm (0.1556 mg/L).

 

n-Butyl acrylate:

 

For nBA an OECD414 in rabbit study is available as well (Charles River 2017).In this study 25 inseminated New Zealand White rabbits were exposed orally (gavage) to 50, 150 and 400 mg/kg bw/day during gestation days 7 through 28. All females in the control, 50, 150, and 400 mg/kg/day groups survived to the scheduled necropsy. No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.A test substance-related absence of a mean body weight gain (0 g) was noted in the 400 mg/kg/day group on the first day of dose administration (gestation day 7-8) and resulted in a 78.9% lower mean body weight gain in this group compared to the control group during gestation days 7-10 and a lower mean body weight gain when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse. In addition, lower mean food consumption was noted in the 400 mg/kg/day group during gestation days 7-10 and resulted in lower mean food consumption in this group compared to the control group when the entire treatment period (gestation days 7-29) was evaluated; however, these differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse. Mean maternal body weights, body weight gains, and food consumption in the 50 and 150 mg/kg/day groups and mean body weights, net body weights, net body weight gains, and gravid uterine weights in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. There were no substance-related macroscopic findings noted at the scheduled necropsy on gestation day 29 in the 50, 150, and 400 mg/kg/day groups. Intrauterine growth and survival in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. In addition, no test substance-related external, visceral, and skeletal malformations or developmental variations were noted at any dosage level.Nonadverse lower mean body weight gains and corresponding lower mean food consumption were noted in the 400 mg/kg/day group. No evidence of developmental toxicity was noted at 50, 150, and 400 mg/kg/day. Based on these results, a dosage level of 400 mg/kg/day, the highest dosage level tested, was considered to be the no‑observed‑adverse‑effect level (NOAEL).

Acrylic Acid:

For investigation of the developmental toxicity of the common metabolite AA groups of 16 pregnant rabbits were exposed (6 h/d, whole-body) to atmospheres containing AA at 0, 25, 75, and 225 ppm (corresponding to approx. 0.075, 0.224, 0.673 mg/L) during days 6-18 of gestation (Bushy Run Research Center, Union Carbide (1993)). All dose groups were observed daily for morbidity and mortality. During the exposure period, animals were observed for clinical signs preceding and subsequent to daily exposures and from outside during actual exposures. Maternal body weights were measured on gestation day 0, 3, 6, 12, 24, and 29. Food consumption was measured daily throughout the study beginning on gestation day 3. After sacrifice on gestation day 29, maternal liver and kidney weights were determined. All foetuses were weighed and examined for external malformations and variations, for thoracic and abdominal visceral abnormalities including internal sex organs, for craniofacial abnormalities and for skeletal malformations and variations. Dose-related clinical signs (as perinasal/perioral wetness and nasal congestion, as well as reduced body weight gain and food consumption) were observed in the 75 and 225 ppm groups. The overall pregnancy rate was equivalent for all groups (94-100 %). No dose-related effects were observed in the reproduction function of the dams. There were no effects on the number of ovarian corpora lutea, the number of total viable or non-viable (early and late resorptions and dead foetuses) implantations/litter. Percentage live foetuses and sex ratio were equivalent across groups. Fetal body weights were unaffected by test substance exposure. There were no exposure-related increases in the incidences of external, visceral or skeletal malformations or variations.NOAEC for maternal toxicity was 25 ppm (= approx. 0.075 mg/L).NOAEC for developmental toxicity: 225 ppm = 0.673 mg/L

 

Ethane-1,2-diol:

 

Administration of ethane-1,2-diol to New Zealand White rabbits (0, 100, 500, 1000, or 2000 mg/kg bw/d; p.o.) caused mortality in 42% of the does as a result of renal failure. In the offspring, no effects upon survival, foetal weight, or the incidences of external, visceral, or skeletal malformations or variations were observed at any dose level, including the progeny of surviving does treated with 2000 mg/kg/day. Altogether,the substance resulted in profound maternal toxicity at 2000 mg/kg/day (42% mortality; three early deliveries and one spontaneous abortion) associated with renal pathology (Tyl et al. 1993).

 

 

Justification for classification or non-classification

Based on the available data, the test substance does not fulfil the classification as a reprotoxic substance according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.