Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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
Study period:
15 Aug 2018 - 14 Nov 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD guidance document supporting OECD test guideline 443 on the extended onegeneration reproductive toxicity test, No. 151
Version / remarks:
2013
Deviations:
no
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 : 10 weeks

- Basis for dose level selection :
The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422) with oral exposure of the test item in rats. In this preliminary study, animals were dosed with 0, 40, 125 and 375 mg/kg/day. From dose 125 mg/kg/day, adverse findings observed were ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other test item-related findings consisted of an increase in liver weights in males and females and an increase in hyaline droplet accumulation in the male kidneys at 375 mg/kg/day. Based on the severe findings in the stomach, a parental local NOAEL was established of 40 mg/kg/day in this study. As animals are dosed for a longer time period for the Extended One-Generation Reproductive Toxicity Study (e.g. F0-males 11-13 weeks versus 4 weeks in an OECD 422 study), a maximum dose level of 100 mg/kg/day was selected.

- Inclusion/exclusion of extension of Cohort 1B : Inclusion

- Termination time for F2 : not applicable

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : Exclusion

- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : Exclusion

- Route of administration : The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Test material

Constituent 1
Chemical structure
Reference substance name:
(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate
EC Number:
256-032-2
EC Name:
(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate
Cas Number:
42978-66-5
Molecular formula:
C15 H24 O6
IUPAC Name:
(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6-7 week; (F1) 3 wks
- Weight at study initiation: (P) Males: 136 and 187 g; Females: 112 and 153 g; (F1) Males: mean 50 g; Females: 49 g
- Fasting period before study: During motor activity measurements, F0- female animals had no access to food for a maximum of 2 hours. During motor activity measurements, F0-female animals had no access to water for a maximum of 2 hours.
- Housing:
On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III)
During the post-mating phase, males were housed in their home cage (Macrolon type IV) with a maximum of 5 males/cage). Females were individually housed in Macrolon plastic cages (type III).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height). Pups were housed with the dam until termination or weaning (on PND 21).
During locomotor activity monitoring, F0-females were housed individually in a Hi-temp polycarbonate cage
- Diet: ad libitum, Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, Municipal tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 45 to 67
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least weekly as a solution, formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 2, 6, 20 mg/mL
- Amount of vehicle: 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (type III).
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulations. The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
see table 1
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
25 (F0) / 20 (F1)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422) with oral exposure of the test item in rats and in an attempt to produce graded responses to the test item. In this preliminary study, aninmals were dosed with 0, 40, 125 and 375 mg/kg bw/day. From dose 125 mg/kg bw/day, adverse findings observed were ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other test item-related findings consisted of an increase in liver weights in males and females and an increase in hyaline droplet
accumulation in the male kidneys at 375 mg/kg/day. As animals are dosed for a longer time period for the Extended One-Generation Reproductive Toxicity Study (e.g. F0-males 11-13 weeks versus 4 weeks in an OECD 422 study), a maximum dose level of 100 mg/kg bw/day was selected. No (severe) clinical symptoms and effects on body weight and food consumption were noted (i.e. the general health of the animals was good). The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.


Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION AND COMPOUND INTAKE: No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL TESTS – F0-Generation
Functional tests were performed on the selected 5 females during Week 10 of treatment. The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity

CLINICAL PATHOLOGY - F0-Generation and Cohort 1A animals
Sample collection:
Blood of 10 selected animals/sex/group of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy. Samples were collected from the retro-orbital sinus under anaesthesia using isoflurane in the animal facility.
The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0-animals and Cohort 1A animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.
Oestrous cyclicity (parental animals):
Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken.
Sperm parameters (parental animals):
Parameters examined in [F0, Cohort 1A] male parental generations:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing, the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
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
The following parameters were examined in [F1] offspring:

F1-Generation until Weaning (PND 21):

Mortality/Moribundity Checks
Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.

Body Weights
Live pups were weighed individually on PND 1, 4, 7, 13 and 21. For animals of Cohort Surplus, a terminal weight was recorded on the day of scheduled necropsy.

Sex
Sex was externally determined for all pups on PND 1 and 4 (sex determination not noted for Day 13)

Anogenital Distance
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

Areola/Nipple Retention
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Clinical Pathology
On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation for determination of thyroid hormone.

F1-Generation from Weaning (PND 21) onwards:

Mortality/Moribundity Checks – Cohorts 1A, 1B and 1C
Throughout the study, animals were observed for general health/mortality and moribundity twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations– Cohorts 1A, 1B and 1C
Clinical observations were performed at least twice daily, up to the day prior to necropsy. These observations were at least conducted prior to dosing and 0-30 minutes after dosing.

Body Weights – Cohorts 1A, 1B and 1C
Animals were weekly weighed individually. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. For animals of Cohorts 1A and 1B, a terminal weight was recorded on the day of scheduled necropsy.

Food Consumption– Cohorts 1A, 1B and 1C
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.

Water Consumption – Cohorts 1A, 1B and 1C
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Vaginal Patency – Cohorts 1A, 1B and 1C
Vaginal patency (vaginal opening) was monitored daily for all females from PND 25 onwards until vaginal patency was present, by visual inspection of the vaginal area.

Balanopreputial Separation – Cohorts 1A, 1B and 1C
Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area.

Stage of Estrus Determination – Cohorts 1A, 1B and 1C
Estrous stages were determined by examining the cytology of vaginal lavage sample, taken on the day of scheduled necropsy.

Estrous Cycle Determination – Cohort 1A
Estrous stages were determined by examining the cytology of vaginal lavage samples, taken during two periods. During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.

Clinical Pathology - F1- animals of Cohort Surplus on PND 22
On PND 22, blood was collected from all Cohort Surplus animals (10/sex/group), if possible for determination of Thyroid Hormone. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.

Clinical Pathology – Cohort 1A
Blood of 10 selected animals/sex/group of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy for
- Hematology: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelets, Reticulocytes (absolute), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- Clinical Chemistry: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT),Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos)
- Thyroid hormone: Thyroxine (T4), Thyroid Stimulating Hormone (TSH)
Urinalysis: Specific gravity, White blood cells (WBC-sed.), Clarity Red blood cells (RBC-sed.), Colour Casts, pH, Epithelial cells, Blood Crystals, White blood cells (WBC), Bacteria, Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite

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

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: yes
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. If possible, one pup (male or female) was selected per litter (20 litters in total). One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 μm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The following subpopulations were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy:
T-cells, T-helper cells, T-cytotoxic cells, B-cells, NK-cells, Ratio T-helper cells/ T-cytotoxic cells (Th/Tc)
The % lymphoid cells of peripheral blood mononuclear cells (PBMC) were determined using the Forward Scatter and Side Scatter.

Postmortem examinations (parental animals):
SACRIFICE- F0 Generation
Animals surviving until scheduled euthanasia were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies:
Males (which sired and failed to sire): After successful mating and a minimum of 10 weeks of treatment.
Females which delivered: LD 23-25.
Females which failed to deliver:
With evidence of mating: Post-coitum Days 25-27 (102,105, 108, 111, 122, 144, 179, 194, 200).
Without evidence of mating: 24 days after the last day of the mating period (Nos. 112, 168)
Females with total litter loss (No. 126): Within 24 hours after the last pup was found dead or missing.

Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy. Water was available.

GROSS NECROPSY- F0 Generation
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no
macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS - F0 Generation
The organs identified in Table 5 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios were calculated. Representative samples of the tissues identified in Table 5 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.
Postmortem examinations (offspring):
SACRIFICE -F1 Generation until weaning
Unscheduled Deaths– F1-Generation
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible). Descriptions of all external abnormalities were recorded. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. The
stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Culled Pups (PND 4) – F1-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation.

GROSS NECROPSY
See table 3 for details

Cohort 1A
Scheduled necropsy of Cohort 1A was conducted on PND 89-95. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

Cohort 1B
Scheduled necropsy of Cohort 1B was conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort 1C
Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy and no terminal body weight was recorded. All
animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort Surplus
Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGTHS
Cohort 1A
In addition to the procedures described above, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A animals of Group 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea.
Cohort 1B
In addition to the procedures described above, the reproductive organs of all Cohort 1B animals were processed to block stage.
Statistics:
Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The motor activity data set (at least 3 groups) was compared using an overall Kruskal-Wallis.
Incidence:
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
see table 4
Offspring viability indices:
see table 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test-item related clinical signs were noted up to treatment with 30 mg/kg bw/day. No findings were noted during the weekly arena observations in this study.
Salivation seen after dosing among animals of the 100 mg/kg bw/day dose group during the first 8 weeks of the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than being a sign of systemic toxicity. Other clinical signs noted during the treatment period occurred within the range of
background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No test item-related mortality occurred during the study period. One female of the 10 mg/kg bw/day group was euthanized on Lactation Day 1, as she had a total litter loss.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered to have been unaffected by treatment up to 100 mg/kg bw/day. A trend towards decreased body weights and body weight gain may appeared in males at 100 mg/kg bw/day up to Week 1 of the mating period. A statistical significant decrease in bodyweight was observed at 100 mg/kg bw/day in Week 8 of treatment. The statistical significant increase in body weight gain at 30 mg/kg bw/day in Week 3 of mating was considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight in animals treated up to 100 mg/kg bw/day was similar to the control level over the treatment period. Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in haematological parameters. Statistically significant lower white blood cell count (WBC) was noted in females at 10 and 100 mg/kg bw/day, which was likely the result of lower lymphocytes levels observed in these animals. In absence of a dose response and as values remained within normal range, these changes were considered unrelated to treatment with the test item. Any other statistically significant changes in haematology parameters achieving a level of statistical significance when compared to controls were considered unrelated to administration of the test item due to the minimal magnitude of the change and/or absence of a dose response. Coagulation parameters of treated rats were considered not to have been affected by treatment up to 100 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were observed in treated males at 30 and/or 100 mg/kg bw/day:
Increase in urea at 100 mg/kg/day (1.16x, 4.3 mmol/L).
• Increase in sodium at 30 and 100 mg/kg bw/day (1.01x (143.3 mmol/L) and 1.02x (143.8 mmol/L), respectively).
• Increase in potassium at 100 mg/kg bw/day (1.07x, 4.02 mmol/L)
Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment with the test item. Serum levels of TSH and T4 in F0-males and -females were not affected by treatment up to 100 mg/kg bw/kg.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis was not affected by treatment with the test item up to 100 mg/kg bw/day.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. Motor activity (total movements and ambulations) was (non-statistically) slightly increased in high dose animals. This slight increase was considered due to one female, for which a level of total movements and ambulations was recorded.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic alterations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Histopathological examination of reproductive tissues revealed no evidence of a treatment-related effect on reproduction.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were unaffected by treatment with the test item. All females had regular cycles of 4 to 5 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm motility, concentration and morphology parameters were unaffected by treatment up to 100 mg/kg bw/day. Statistically significant changes noted were considered unrelated to treatment as a dose-related response was absent, and since the opposite effect would be expected in case of toxicity.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating index was considered not to be affected by treatment. With the exception of one control and one 30 mg/kg bw/day female, all females showed evidence of mating. Precoital time was considered not to be affected by treatment. With the exception of one female (mated at Day 14), all females showed evidence of mating within the first 4 days. Number of implantation sites was considered not to be affected by treatment up to 100 mg/kg bw/day. Fertility index was considered not to be affected by treatment. The fertility indices were 79%, 96%, 100% and 88% for the control, 10, 30 and 100 mg/kg bw/day groups, respectively. A total of 5 control females, one female at 10 mg/kg bw/day and 3 females at 100 mg/kg bw/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: A dose level of 100 mg/kg bw/day was selected as the NOAEL of 40 mg/kg bw/day was established in a dose range finder. Here, adverse findings consisted of ulcers and inflammation of the forestomach and hyperplasia squamous cells from 125 mg/kg bw/day on.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest doses tesed

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):
No clinical signs occurred among pups that were considered to be related to treatment. For the pup of one Female (10 mg/kg bw/day) which was missing on Day 2, absence of milk in the stomach was noted on Day 1 and for the pup of Female No. 154 which was missing on Day 2, a pale appearance was noted on Day 1. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant. No test item-related clinical signs were noted during daily detailed clinical observations in Cohort 1A,1B and 1C or during weekly arena observations.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No mortality occurred during the study period in Cohort 1A, 1B and 1C from weaning onwards that was considered to be related to treatment with the test item.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment.
Body weights and body weight gains of Cohort 1A, 1B and 1C were considered unaffected by treatment up to 100 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before and after correction for body weight was considered unaffected by treatment up to 100 mg/kg bw/day.
Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment as changes were only minimal and/or no trend was apparent regarding dose and duration of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: The following statistically significant changes were noted in male animals at 100 mg/kg bw/day.
• Increased neutrophil levels (1.3x)
• Increased lymphocyte levels (1.16x)
The statistically significant change in white blood cell count (WBC) in males at 10 mg/kg bw/day was considered not to be related to treatment with the test item as it occurred in the absence of a dose-related trend. Coagulation parameters of treated rats were considered not affected by treatment up to 100 mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female pups, culled at PND 4 and male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment. Serum TSH levels in male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment.
Cohort 1A: Clinical chemistry parameters of treated rats were considered not affected by treatment up to 100 mg/kg bw/day. Serum levels of thyroid stimulating hormone (TSH) and Total T4 were considered not to be affected by treatment up to 100 mg/kg bw/day.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Cohort 1A: Urinary parameters were unaffected by treatment.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
Balanopreputial separation and vaginal patency were considered unaffected by treatment up to 100 mg/kg bw/day. At 30 mg/kg bw/day, an increase in the age of reaching balanopreputial separation was observed (1.03x compared with control). This was likely related to Male No. 355 that had a very low body weight and had reached balanopreputial separation at age PND 54 (versus average age of reaching balanopreputial separation on PND 41.5 for control animals).
At 10 mg/kg/day, an increase in age of reaching first estrus (1.04x compared with controls) was noted in females. In absence of a dose response, this slight increase was considered unrelated to treatment.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 100 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related organ weight changes in the Cohort surplus-animals.

Cohort 1A and 1B:There were no test item-related organ weight changes observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment.
Cohort 1A and 1B: There were no test item-related macroscopic alterations in the F1-generation. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in several females in all dose groups in Cohort 1A and 1B, is related to a stage in the estrous cycle and is a normal finding.
Histopathological findings:
no effects observed
Description (incidence and severity):
Cohort 1A: There were no test item-related microscopic alterations in Cohort 1A of the F1-generation.. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Spermatogenesis-staging (Cohort 1A)
Stage-dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Ovarian Follicle Counts - Cohort 1A
There were no test item-related effects on the ovarian follicle counts in the F1-females (Cohort 1A) at 100 mg/kg/day when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous Cycle – F1-Generation (Cohort 1A):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had regular cycles of 4 to 5 days. Female No. 636 (30 mg/kg bw/day) had two cycles with a duration of 2 days and one of 5 days and was therefore classified as having an irregular cycle. For Female Nos. 495 (control) and 555 (10 mg/kg bw/day) estrous cycle regularity/length could not be determined. Considering the low incidence and in absence of a dose response, this finding was considered unrelated to treatment with the test item.
Sperm Analysis – F1-Generation (Cohort 1A):
At 100 mg/kg bw/day, an increase in spermcount of the epididymides was observed of 1.23x compared with controls. This was considered unrelated to treatment as a dose-related response was absent, and since the opposite effect would be expected in case of toxicity.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Cohort 1A: There were no test item-related effects on splenic lymphocyte subpopulations observed up to 100 mg/kg bw/day.
Higher T-cell and T-cytotoxic cell splenic subpopulations and lower B-cell splenic subpopulations observed for females reached statistical significance when compared with controls at 10 mg/kg bw/day. However, these shifts were slight of nature and occurred in the absence of a dose-related response and in absence of any test item-related microscopic findings in the spleen. As such, these shifts were considered to represent biological variability and were considered not to be related to treatment.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
A study according OECD TG 443 with the read-across substance TPGDA (CAS 42978-66-5) (ReachCentrum, 2019C) is available which was conducted to provide an evaluation of possible pre- and postnatal effects on development. In addition, a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats was performed. Based on the results of the extended one generation reproductive toxicity study (ReachCentrum, 2019C) with the read-across substance TPGDA (1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate (CAS 42978-66-5), the following no-observed-adverse-effect levels (NOAEL) were established for the read-across substance:
General toxicity (F0 and F1): at least 100 mg/kg bw/day
Reproduction: at least 100 mg/kg bw/day
Developmental: at least 100 mg/kg bw/day
Executive summary:

An oral (gavage) study according OECD TG 443 with the read-across substance TPGDA (CAS 42978-66-5) (ReachCentrum, 2019C) is available which was conducted to provide an evaluation of possible pre- and postnatal effects on development. In addition, a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats was performed. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of the substance on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

This Extended One Generation Reproductive Toxicity Study included Cohort 1 for assessment of reproductive/developmental toxicity. Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 10, 30 and 100 mg/kg bw/day. The animals of the control group received the vehicle, Corn Oil, alone. F0-males were treated for 11-13 weeks, including 10 weeks prior to mating and during the mating and post-mating period, up to and including the day before scheduled necropsy. F0-females were treated for a minimum of 14-18 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 14 weeks. From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B and 1C, were dosed up to and including the day before scheduled necropsy (i.e. for a minimum of 3 weeks). The F1-animals of Cohort Surplus and Spares (not assigned to one of the cohorts) were not dosed. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

F0-Generation: No parental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). At 30 and 100 mg/kg bw/day, a minor increase in sodium was noted in males and additionally, at 100 mg/kg bw/day, urea and potassium levels were slightly increased. As values were only slightly above the range considered normal (sodium) or remained within normal range (urea and potassium) and no corroborative microscopic findings were found, these changes were considered non-adverse. No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (females), body weight, food consumption, haematology, coagulation, thyroid hormone analysis, urinalysis, macroscopic examination, organ weights and microscopic examination).

Reproductive results – F0-generation

No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). No test item-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, sperm analysis, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Developmental results – F0-generation / F1-generation (pre-weaning)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day) during the pre-weaning phase. No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, duration of gestation, viability and weaning indices, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), and macroscopic examination).

Developmental results – F1 generation (post-weaning)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day) during the post-weaning phase. At 100 mg/kg bw/day, increases in neutrophil and lymphocyte levels were found in males. As values remained within normal range and no correlative microscopic findings were found, these changes were considered non-adverse. No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. mortality, clinical signs, body weight, food consumption, coagulation, clinical chemistry, thyroid hormone analysis, urinalysis, splenic lymphocyte subpopulation, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrous, time between vaginal opening and first estrous length and regularity of the estrous cycle, sperm analysis, macroscopic examination, organ weights, ovarian follicle and corpora lutea counts, and microscopic examination, including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Higher doses could not be tested as these were not tolerated (according to the Dose-Range Finder Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test).

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1), the following no-observed-adverse-effect levels (NOAEL) of the read across substance were established:

General toxicity (F0 and F1): at least 100 mg/kg bw/day

Reproduction: at least 100 mg/kg bw/day

Developmental: at least 100 mg/kg bw/day

A maximum dose level of 100 mg/kg bw/day was selected as a NOAEL of 40 mg/kg bw/day was established in a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test with oral exposure of the read across substance in rats. In this preliminary study, adverse findings consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females from 125 mg/kg bw/day.