Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxyethyl acrylate
EC Number:
212-454-9
EC Name:
2-hydroxyethyl acrylate
Cas Number:
818-61-1
Molecular formula:
C5H8O3
IUPAC Name:
2-hydroxyethyl acrylate
Test material form:
liquid
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)

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Effect levels (P0)

open allclose all
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:
12 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

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.

Applicant's summary and conclusion