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Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed on 2 moles ethoxylated bisphenol A dimethacrylate by oral gavage in male and female Wistar Han rats. Animals were treated with dose levels of 62, 250 and 1000 mg/kg revealed no parental toxicity or developmental/reproductive toxicity up to 1000 mg/kg.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Augustus - 09 November 2012

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

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Version / remarks:

2000

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: Charles River Deutschland, Sulzfeld, Germany
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 306-309 gr (males) or 213 - 220 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES
From: 23 Augustus to 09 November 2013

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance and the vehicle. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating.
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
-Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The delegated phase was performed by the Principal Investigator for Formulation Analysis.

Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands.

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Formulation samples were directly shipped instead of being sent to the ‘weegkamer’ first.
Evaluation: The test site received the samples in good condition; the study was not affected.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to the scheduled necropsy. Females were exposed for 43-46 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).Three females( from Group 1, 2 and 3 respectively) and 2 females from Group 4 were not dosed during littering.

Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 d/w.

Details on study schedule:

- Age at mating of the animals in the study: Approximately 13 weeks

Dose / conc.:

62 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 14-Day dose range finding study (See attached results).
- Based on the results of this range finding study, dose levels for the main study were: 62, 250 and 1000 mg/kg.

- Since no clinical signs were observed in the range finding study, clinical observations in the main study were started at no specific time point but within a similar time period for all animals after dosing.

- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Positive control:

Not required.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were conducted for all animals at no specific time point, but within a similar time period after dosing for the respective animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: yes

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

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
- Time schedule for collection of blood: prior to scheduled post mortem examination (between 7.00 and 10.30 a.m.).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY
- Time schedule for collection of blood: prior to scheduled post mortem examination (between 7.00 and 10.30 a.m.).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

THYROID HORMONE ANALYSIS
- Time schedule for collection of blood: prior to scheduled post mortem examination (between 7.00 and 10.30 a.m.).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines
- Instead of taking 1 mL of blood for the serum sample from males, 0.5 mL was initially taken and an additional 0.5 mL had to be collected again. Animals. 3 and 5 (Group 1) had already been necropsied, so an additional 0.5 mL could not be collected.
Evaluation: The extra collection has no effect on the study and sufficient data is available for the insufficient samples from animals 3 and 5.

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines
- Motor activity and functional observation assessment were performed on Day 3 of lactation for one Group 4 animal instead of from Day 4.
Evaluation: Conducting these tests one day too early has no impact on the results.

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group and animals suspected to be infertile).

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
During the lactation period, no clinical observations were entered online for pups 13 -15 of 1 Group 2 litter on Day 6 and for pups 13 +14 of an other Group 2 on Day 5.
Evaluation: Sufficient data is available for a thorough evaluation.

- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
All animals were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- The number of former implantation sites and corpora lutea were recorded for all paired females.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining animals and females which failed to deliver: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
- All remaining males: Epididymides and testes

- Inadvertently, the testes and epididymides from 1 Group 2 animal were not weighed at necropsy, and the rectum from 1 Group 1 animal and the mesenteric lymph node from 1 Group 4 animal were not available for histopathology because the tissues were not discernible at necropsy or trimming, or were erroneously not collected.
Evaluation: Sufficient data was available for evaluation.

HISTOPATHOLOGY
- According to test guidelines
- The skin was collected from all Group 1 and 2 animals at necropsy.
Evaluation: The tissue was stored but not analyzed further. This has no influence on the study.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100
- Pre-implantation loss (%): (number of corpora lutea - number of implantation sites)/number of corpora lutea x 100
- Post-implantation loss (%): (number of implantation sites - number of live fetuses) / number of implantation sites x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No adverse clinical signs of toxicity were noted during the observation period.
Salivation was seen for all animals at 1000 mg/kg, for four males and one female at 250 mg/kg and for eight males at 62 mg/kg. This was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related taste or possible irritancy of the test substance, and is not reflective of systemic toxicity.
Incidental findings noted included chromodacryorrhea and opacity of the right eye, noted for a single control male. These findings 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. As these were noted for a control male, they were not treatment related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One Group 4 female no. 79 was found dead the morning after pairing began. She was partially cannibalized with her vagina, urinary bladder, rectum and clitoral glands missing. Severe necrosis of the trachea was found at the microscopic examination, indicating her death was secondary to a gavage error and was not attributable to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg.
Body weight gain was slightly lower for males at 250 and 1000 mg/kg on Day 8 of the premating period. The difference from controls was only very slight and moreover, no statistically significant decrease in body weight for males was observed during the premating period for males. Taken together, it was not considered to be toxicologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically relevant effects on haematology parameters up to 1000 mg/kg.
The significant increase in mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) seen for males at 62 and 250 mg/kg. These parameters were unaffected for females.
The significant decrease in eosinophils was seen for females at 250 mg/kg only. A trend towards an increase in eosinophils with increasing doses was seen for males, but changes were not statistically significant. All these changes occurred in the absence of a dose response effect and remained within the range considered normal for animals of this age and strain. As such, these differences from controls were not considered to be toxicologically relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cholesterol was significantly higher than controls for males at 250 and 1000 mg/kg and for females in all treated groups. Both sexes show a clear dose response effect. However, in the absence of toxicologically significant findings in any other parameters, it was not considered to be toxicologically relevant.
The significant decrease in albumin was noted for males (all treated groups); the same decrease dose-related decrease was seen for females, but was not statistically significant.
The significant decrease in creatinine seen for females (62 and 250 mg/kg) did not show a dose-dependent trend as females at 1000 mg/kg were not affected. There were no creatinine changes for males. Therefore, this change was not considered to be toxicologically relevant as it occurred in the absence of a treatment related distribution and remained within the range considered normal for rats of this age and strain.
Males at 62 mg/kg had a significant increase in the thyroid hormone total thyroxine (T4) compared to controls. This was also not considered to be toxicologically relevant as it occurred in the absence of a treatment related distribution and no corroborative effects were seen microscopically.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.
The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

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 microscopic findings were found in female thyroid glands and comprised:
Diffuse follicular hypertrophy/hyperplasia that was seen in 2/5 males (minimal) and 1/5 females (slight) of Group 2, 2/5 males (minimal) of Group 3 and 1/5 males (slight) and 3/5 females (minimal) of Group 4. However, this remained within the normal background range of findings for rats of this age and strain, and based on the absence of a dose-relationship, the diffuse follicular hypertrophy/hyperplasia was considered to be adaptive in nature and not toxicologically relevant.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.
All microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain and in this type of study.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. There were no toxicologically relevant effects on the gestation index and duration, parturition or maternal care. There were 9, 10, 10 and 9 pregnant females in the control, 62, 250 and 1000 mg/kg groups, respectively.
When recalculated without counting the single implantation site for one control female, the mean number of implantation sites for controls was 13.3. Even discounting this female, there were no differences between control and treated females.
For one Group 3 female, the number of pups (14) was slightly higher than the number of implantations (13). This was considered to be caused by normal resorption of implantation sites as these enumerations were performed on Day 7 of lactation
The gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg.
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed. There were 8, 10, 10 and 9 females with living pups on Day 1 of lactation. One Group 1 female (control) was found with one giant fetus in the uterus at the macroscopic examination, where the fetus was so large it would not be possible for the mother to deliver. One Group 4 Female (1000 mg/kg) was found dead the day after the mating period began.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental clinical signs of pups included pale appearance and blue spot of the abdomen, which were noted for a single pup at 250 mg/kg. The nature and incidence of these signs remained within the range considered normal for pups of this age, and were not considered toxicologically relevant. No clinical signs were noted for any other pup.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Two pups (from separate litters) at 1000 mg/kg were missing on Day 2. They were most likely cannibalized. There were no differences in Day 1 body weights between these pups and control pups. No toxicological relevance was attributed to these dead/missing pups since the incidence remained within the range considered normal for pups of this age. There were no other pups that died or went missing in the control or other treated groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were unaffected by treatment up to 1000 mg/kg.

Food consumption and compound intake (if feeding study):

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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The only macroscopic finding noted was blue spot on the abdomen, noted for a single pup at 250 mg/kg. Due to the nature of this finding and its limited incidence, it was not considered to be toxicologically relevant.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

No test substance was detected in the Group 1 formulations from the week 1, 3 and 6 preparations.

 

The concentrations analyzed in the formulations of Groups 2, 3 and 4 in weeks 1, 3 and 6 were in

agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

 

The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation = 10%).

Formulations over the entire range were stable when stored at room temperature under normal

laboratory light conditions for at least 5 hours (i.e. relative difference = 10%).

 

The long term storage samples were stable at =-70°C for at least 21 days.

Conclusions:

In conclusion, treatment with 2 moles ethoxylated bisphenol A dimethacrylate by oral gavage in male and female Wistar Han rats at dose levels of 62, 250 and 1000 mg/kg revealed no parental, developmental or reproductive toxicity up to 1000 mg/kg.

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

Executive summary:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats with 2 moles ethoxylated bisphenol A dimethacrylate by oral gavage (OECD guideline 422).

Based on the results of a 14-day dose range finding study, the dose levels for this study were 62, 250 and 1000 mg/kg. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-46 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily),

functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

 

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg). No toxicologically relevant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg).

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

No treatment-related changes were noted in any reproductive or developmental parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers ofcorpora lutea and implantation sites, gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Treatment with 2 moles ethoxylated bisphenol A dimethacrylate by oral gavage in male and female Wistar Han rats at dose levels of 62, 250 and 1000 mg/kg revealed no parental, developmental or reproductive toxicity up to 1000 mg/kg. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

WIL study is a reliable study, performed according to an OECD guideline study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screeningtest (WIL 2013):

This study was performed in rats with 2 moles ethoxylated bisphenol A dimethacrylate byoral gavage (OECD guideline 422).

Based on the results of a 14-day dose range finding study, the dose levels for this study were 62, 250 and 1000 mg/kg. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-46 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily),

functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

 

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg). No toxicologically relevant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg).

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

No treatment-related changes were noted in any reproductive or developmental parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers ofcorpora lutea and implantation sites, gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Treatment with 2 moles ethoxylated bisphenol A dimethacrylate by oral gavage in male and female Wistar Han rats at dose levels of 62, 250 and 1000 mg/kg revealed no parental, developmental or reproductive toxicity up to 1000 mg/kg. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

Effects on developmental toxicity

Description of key information

A developmental toxicity study in rat was performed on 2 moles ethoxylated bisphenol A dimethacrylate.

No adverse effect was observed in parental females or in foetus developmental at the highest dose of 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2017 - 20 July 2017

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 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France
- Age: at the beginning of the treatment period, the animals were 10-11 weeks old
- Mean body weight: at the beginning of the treatment period, the animals had a mean body weight of 300 g (range: 255 g to 360 g)
- Fasting period before study: no
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 4 or 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): aboutr 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 26 June 2017 to 20 July 2017.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection (HPLC/UV)
Test item concentrations: remained within an acceptable range of variations (-1.8% to +8.6%) when compared to the nominal values (± 15% of the nominal concentrations)
Homogeneity: the dose formulations containing the test item and prepared at 2 mg/mL and 200 mg/mL in PEG 400 were found to be homogeneous after preparation. Dose formulations ranging from 2 mg/mL to 200 mg/mL are therefore considered to be suitable for routine administration in GLP Toxicological studies, and have to be maintained under magnetic agitation after preparation and during the administration procedure to animals in follow-up studies.
Stability: not assessed, dose formulation prepared daily

Details on mating procedure:

The females were mated at the breeder's facility. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c.

Duration of treatment / exposure:

The dose formulations were administered daily from Day 6 to Day 20 p.c. inclusive.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor, based on the results of a previous OECD 422 study performed in Wistar Han rats.
In the previous study, rats received the test item daily by gavage at dose levels of 62, 250 or 1000 mg/kg/day for 29 days (males) or 43 to 46 days (females).
There were no toxicologically relevant changes in any of the parental parameters (i.e. clinical signs, Functional Observation Battery, motor activity, body weight, body weight change, food consumption, clinical laboratory investigations and histopathology examination). Mating, fertility and conception indices, pre-coital time, numbers of corpora lutea and implantation sites, gestation index, duration of gestation, and pup body weights were unaffected by the treatment and no developmental toxicity was observed up to the dose level of 1000 mg/kg/day.
Based on these available data, the dose levels selected for the present study were 100, 300 and 1000 mg/kg/day.

- Rationale for animal assignment: computerized stratification procedure.

Maternal examinations:

MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL SIGNS:
- Time schedule: from arrival, each animal was observed once a day as part of the routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 21 post-coitum.
- Examined: principal thoracic and abdominal organs.

Ovaries and uterine content:

The ovaries and uterus of the females were examined to determine:
- gravid uterus weight,
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites,
- gross evaluation of placentas.

Fetal examinations:

- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: half fetuses per litter
- Skeletal examinations: Yes: half fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other: number dead and live, body weight, sex.

Statistics:

Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by the Fisher exact probability test (proportions).

Indices:

% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Historical control data:

Cf attached document

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See table 1.
Test item-related clinical signs consisted of ptyalism, which was observed for 1 to 11 days in many females given 100 mg/kg/day and in most females given 300 or 1000 mg/kg/day. These clinical signs were considered as non-adverse.
Emaciated appearance (without body weight loss), associated with piloerection, was noted in 1/24 females given 100 mg/kg/day from Day 20 p.c. These findings were considered to be unrelated to the test item treatment, as they were not dose-related and were reported in only one animal.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See table 2.
There were no effects on mean body weight or mean body weight change.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See table 3.
There were no effects of treatment with the test item.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

See table 4.
There were no test item-related macroscopic findings.
The macroscopic observations were not attributed to the test item treatment as they were reported with only isolated incidences, were not dose-related and/or are findings commonly observed in rats of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

See table 5.
There were no test item-related effects on mean gravid uterus weight, mean carcass weight or net body weight change.
At 1000 mg/kg/day females had a low mean net body weight change when compared with controls (-15%). This was mainly due to the high value recorded in one control female (one female: +117 g). This difference, which was not statistically significant and which did not correlate with any other findings, was therefore considered not to be test item-related.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

See table 6.
There were no test item-related effects.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

See table 6.
There were no test item-related effects.

Early or late resorptions:

no effects observed

Description (incidence and severity):

See table 6.
There were no test item-related effects.

Dead fetuses:

no effects observed

Description (incidence and severity):

See table 6.
No dead fetuses.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

See table 6.
There were no test item-related effects.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

See table 7.
There were no effects on mean fetal body weight

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

See table 7.
There were no effects on sex ratio (percentage of male fetuses).

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

See table 8 (variations)
There were no external variations in the test item-treated groups.
In the control group, one fetus had protruding tongue; this finding was within the range of the historical control data.


See table 9 (malformations)
There was no test item-related increase in the frequency of external malformations.
In the 1000 mg/kg/day group, there was one malformed fetus (with anasarca). As anasarca was observed with a similar litter incidence in the control group, it was considered to be unrelated to the test item treatment.
In the 100 and 300 mg/kg/day groups, one litter per dose level had a malformed fetus (2 litters with omphalocele, respectively). As this malformation was observed without a dose-relationship and was within the range of the historical control data, it was considered to be unrelated to the test item treatment.
In the control group, one fetus had anasarca and omphalocele; these findings are commonly observed in this species and strain (Morita et al., 1987).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

See table 10 (cartilage)
There were no relevant differences from controls in the frequency of cartilage observations.
In the 1000 mg/kg/day group and when compared with controls, there was a slight increase in presence of sternebra(e) cartilage. As the fetal and litter incidences were of low magnitude and within or very close to the range of the historical control data, these effects were considered to be of no toxicologically importance (see § Variations).
The other findings were observed in control and treated groups, without a dose relationship and/or with an isolated incidence and/or with an incidence similar to the historical control data. Therefore they were considered to be unrelated to the test item treatment.

See table 11 (variations)
There were no relevant differences from controls in the frequency of skeletal variations.
In the 1000 mg/kg/day group and when compared with controls, there was a slight increase in skeletal variations (i.e. incomplete ossification of 6th sternebra and/or extra external ossification site). As the fetal and litter incidences were of low magnitude and within or very close to the range of the historical control data, these test item-related effects were considered to be of no toxicologically importance in absence of test item-related effects on mean fetal body weight.
The other variations [i.e. incomplete ossification of interparietal, incomplete ossification of thoracic vertebra(e) centrum and incomplete ossification of caudal vertebra(e) arch] were observed in control and/or treated groups, without a dose relationship and/or with an isolated incidence and/or with an incidence similar to the historical control data. Therefore they were considered to be unrelated to the test item treatment although the incidences were sometimes statistically significant.

See table 12 (malformations)
There was no test item-related increase in the frequency of skeletal malformations.
In the 1000 mg/kg/day group, one litter had a malformed fetus (with split interparietal) and one other litter had two malformed fetuses (with less than 15 caudal vertebrae). As these malformations were of isolated occurrence and/or as fetal and litter incidences were within or close to the historical control data, a test item-related effect was considered to be unlikely.
In the 300 mg/kg/day group, one litter had a malformed fetus (with split interparietal). As the malformation was of isolated occurrence, a test item-related effect was considered to be unlikely.
In the 100 mg/kg/day group, one litter had a malformed fetus (with fused ribs). As this malformation was observed without a dose-relationship and was within the range of the historical control data, it was considered to be unrelated to the test item treatment.
In the control group, two litters had one malformed fetus (one with an extra sternebra, and one with absent digits).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

See table 13 (variations)
There was no test item-related increase of the frequency of soft tissue variations.
Tissue variations (i.e. irregular color of the liver, colored focus on the liver, short or absent innominate artery, dilated renal pelvis, dilated ureter, fluid-filled abdomen and/or fluid-filled thoracic cavity) were observed in control and/or treated groups, without a dose-relationship, with an isolated incidence and/or with an incidence similar to the historical control data. Therefore they were considered to be unrelated to the test item treatment.

See table 14 (malformations)
There was no test item-related increase in the frequency of soft tissue malformations.
In the 1000 mg/kg/day group, one litter had a malformed fetus with absence of the left kidney and the left ureter.
In the 300 mg/kg/day group, one litter had a malformed fetus with marked dilated renal pelvis and marked dilated ureter on the right side.
In the 100 mg/kg/day group, one litter had a malformed fetus with marked dilated renal pelvis and marked dilated ureter on the right side.
In the control group, one litter had a malformed fetus with marked dilated renal pelvis.
These tissue malformations were observed in control and treated groups, without a dose relationship, with an isolated incidence and/or with an incidence similar to the historical control data. Therefore they were considered to be unrelated to the test item treatment.

Other effects:

not specified

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Clinical signs

 

Dose level (mg/kg/day)	0	100	300	1000
Ptyalism		13	22	21
Piloerection		1
Emaciated appearance		1
Number of affected animals	0/24	14/24	22/24	21/24

 

Table 2: Body weight

 

Dose level (mg/kg/day)	0	100	300	1000
Body weight (g)
Day 6p.c.	302	300	300	299
	-	(-1)	(-1)	(-1)
Day 9p.c.	317	314	315	313
	-	(-1)	(-1)	(-1)
Day 12p.c.	338	335	334	332
	-	(-1)	(-1)	(-2)
Day 15p.c.	354	352	352	351
	-	(-1)	(-1)	(-1)
Day 18p.c.	401	396	395	396
	-	(-1)	(-1)	(-1)
Day 21p.c.	455	449	447	451
	-	(-1)	(-2)	(-1)
Body weight change (g)
Days 6 - 9p.c.	+15	+14	+15	+14
Days 9 - 12p.c.	+21	+21	+19	+19
Days 12 - 15p.c.	+17	+18	+18	+18
Days 15- 18p.c.	+46	+44	+43	+46
Days 18 - 21p.c.	+55	+53	+51	+54
Days 6 - 21p.c.	+153	+149	+147	+152
	-	(-3)	(-4)	(-1)

p.c.: post-coitum.

-: not applicable.

( ): in brackets, percentage differencevs.controls.

Table 3: Food consumption

 

Dose level (mg/kg/day)	0	100	300	1000
. Days 6 - 9p.c.	24	25	25	26
. Days 9 - 12p.c.	27	28	28	29
. Days 12 - 15p.c.	29	29	29	30
. Days 15 - 18p.c.	33	33	33	33
. Days 18 - 21p.c.	34	33	33	34

p.c.:post-coitum

Table 4: Maternal necropsy findings

 

Dose level (mg/kg/day)	0	100	300	1000
Kidney: dilated pelvis, unilateral		1
Kidney, ureter and uterine horn, unilateral absence			1
Ovary: serous cyst(s), unilateral				1
Placenta: enlarged				1 (1 fetus)
Number of affected animals	0/24	1/24	1/24	2/24

 

Table 5: Net body weight change

 

Dose level (mg/kg/day)	0	100	300	1000
Gravid uterus weight	99	99 (0)	98 (-1)	105 (+6)
Carcass weight	356	350 (-2)	349 (-2)	345 (-3)
Net body weight change from Day 6p.c.	+54	+50	+49	+46

( ): in brackets, percentage differencevs.controls.

p.c.:post-coitum.

^(a): weights are rounded values.

Table 6: Hysterectomy data

 

Dose level (mg/kg/day)	0	100	300	1000
Number of pregnant females at hysterectomy	24	23	23	24
Number of females with live fetuses at termination	24	23	23	24
Number of females with total resorption	0	0	0	0
Mean number ofcorpora lutea	15.1	14.7	14.5	15.1
Mean number of implantation sites	13.9	13.4	12.9	14.0
Mean pre-implantation loss (%)	8.5	7.8	10.8	7.3
Mean number of live fetuses	12.4	12.3	12.0	12.8
Dead fetuses (%)	0.0	0.0	0.0	0.0
Mean number of implantation scars	0.0	0.0	0.0	0.0
Mean number of early resorptions	1.3	1.0	0.8	1.1
Mean number of late resorptions	0.2	0.2	0.2	0.2
Mean post-implantation loss (%)	11.4	8.5	7.9	9.0

Table 7: Fetal body weight and sex ratio

 

Dose-level (mg/kg/day)	0	100	300	1000
Mean fetal body weight (g)	5.85 -	5.78 (-1)	5.89 (+1)	5.93(+1)
Mean fetal body weight of males (g)	6.00 -	5.90 (-2)	6.04 (+1)	6.12(+2)
Mean fetal body weight of females (g)	5.65 -	5.66 (0)	5.75 (+2)	5.78(+2)
Mean percentage of male fetuses (%)	49.8	48.4	50.2	49.0

( ): in brackets, percentage differencevs.controls.

-: not applicable.

Table 8: Fetal external variations

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses	24	23	23	24	388
Number of live fetuses	297	283	275	306	5000
Litters affected, n (%)	1 (4.2)	0 (0)	0 (0)	0 (0)	6 (1.5)^(b)
Fetuses affected, n (%)	1 (0.3)	0 (0)	0 (0)	0 (0)	7 (0.1)^(b)
Protruding tongue, F (L)	0.3 (4.2)	0 (0)	0 (0)	0 (0)	0.4 (4.8)^(a)

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

^(a): maximum incidence.

^(b): mean incidence.

Table 9: Fetal external malformations

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses	24	23	23	24	388
Number of live fetuses	297	283	275	306	5000
Litters affected, n (%)	1 (4.2)	1 (4.3)	1 (4.3)	1 (4.2)	11 (2.8)^(b)
Fetuses affected, n (%)	1 (0.3)	1 (0.4)	1 (0.4)	1 (0.3)	13 (0.3)^(b)
Anasarca, F (L) (%)	0.3 (4.2)	0 (0)	0 (0)	0.3 (4.2)	0 (0.0)^(b)
Omphalocele, F (L) (%)	0.3 (4.2)	0.4 (4.3)	0.4 (4.3)	0 (0)	0.4 (5.3)^(a)

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

^(a): maximum incidence.

^(b): mean incidence.

Table 10: Fetal skeletal examinations (cartilage)

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses	24	23	23	24	388
Number of live fetuses	156	147	140	160	2596
Litters affected, n (%)	24 (100.0)	23 (100.0)	22 (95.7)	23 (95.8)	236 (60.8)^(b)
Fetuses affected, n (%)	109 (69.9)	97 (66.0)	82 (58.6)	101 (63.1)	990 (38.1)^(b)
Cartilage of sternebra(e) present, F(L)	1.9 (12.5)	1.4 (8.7)	0 (0)	5.0 (16.7)	3.8 (21.7)^(a)

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

^(a): maximum incidence.

^(b): mean incidence.

Table 11: Fetal skeletal variations

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses	24	23	23	24	388
Number of live fetuses	156	147	140	160	2596
Litters affected, n (%)	24 (100.0)	23 (100.0)	22 (95.7)	23 (95.8)	345 (88.9)^(b)
Fetuses affected, n (%)	112 (71.8)	99 (67.3)	85 (60.7)	104 (65.0)	1267 (48.8)^(b)
6^thsternebra: incomplete ossification, F(L)	0.6 (4.2)	0.7 (4.3)	0 (0)	4.4(16.7)	2.9 (16.7)^(a)
Extra sternebral ossification site, F(L)	3.2 (16.7)	1.4 (8.7)	2.1 (13.0)	3.1 (20.8)	1.7 (10.0)^(a)
Interparietal: incomplete ossification, F(L)	4.5 (20.8)	3.4 (13.0)	1.4 (4.3)	5.6 (16.7)	9.2 (45.0)^(a)
Thoracic vertebra(e): incomplete ossification of centrum, F(L)	0 (0)	3.4* (17.4)*	2.1 (8.7)	1.9 (12.5)	29.6 (87.5)^(a)
Caudal vertebra(e): incomplete ossification of arch, F(L)	0 (0)	0 (0)	0.7 (4.3)	1.3 (4.2)	2.3 (15.0)^(a)

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

^(a): maximum incidence.

^(b): mean incidence.

Statistically significant for number of litters and fetuses: *: p < 0.05

Table 12: Fetal skeletal malformations

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses	24	23	23	24	388
Number of live fetuses	156	147	140	160	2596
Litters affected, n (%)	2 (8.3)	1 (4.3)	1 (4.3)	2 (8.3)	15 (3.9)^(b)
Fetuses affected, n (%)	2 (1.3)	1 (0.7)	1 (0.7)	3 (1.9)	18 (0.7)^(b)
Interparietal: split, F(L)	0 (0)	0 (0)	0.7 (4.3)	0.6 (4.2)	0 (0.0)
Caudal vertebra(e): less than 15 caudal vertebrae, F(L)	0 (0)	0 (0)	0(0)	1.3 (4.2)	0.6 (4.0)^(a)
Extra sternebra, F(L)	0.6 (4.2)	0 (0)	0 (0)	0 (0)	0 (0.0)
Fused ribs F(L)	0 (0)	0.7 (4.3)	0 (0)	0 (0)	0.8 (5.6)^(a)
Hindpaw : absent digit(s), F (L)	0.6 (4.2)	0 (0)	0 (0)	0 (0)	/

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/: not reported in HCD.

^(a): maximum incidence.

^(b): mean incidence.

Table 13: Fetal soft tissue variations

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses	24	23	23	24	387
Number of live fetuses	141	136	135	146	2404
Litters affected, n (%)	6 (25.0)	6 (26.1)	5 (21.7)	3 (12.5)	86 (22.2)^(b)
Fetuses affected, n (%)	9 (6.4)	10 (7.4)	9 (6.7)	4 (2.7)	119 (5.0)^(b)
Liver: irregular color, F(L)	0 (0)	0.7 (4.3)	0 (0)	0 (0)	/
Liver: colored focus, F(L)	0 (0)	1.5 (8.7)	0 (0)	0 (0)	0.8 (5.0)^(a)
Short innominate artery, F (L)	0 (0)	0.7 (4.3)	0 (0)	0.7 (4.2)	3.7 (22.7)^(a)
Absent innominate artery, F (L)	0 (0)	0 (0)	0.7 (4.3)	0 (0)	5.1 (25.0)^(a)
Dilated renal pelvis, F (L)	3.5 (12.5)	2.9 (8.7)	3.7 (13.0)	0.7 (4.2)	9.5 (28.6)^(a)
Dilated ureter, F (L)	6.4 (25.0)	4.4 (13.0)	5.2 (21.7)	1.4 (4.2)	7.1 (28.0)^(a)
Fluid-filled abdomen, F (L)	0 (0)	0 (0)	0 (0)	0.7 (4.2)	0.9 (5.0)^(a)
Fluid-filled thoracic cavity, F(L)	0 (0)	0 (0)	0 (0)	0.7 (4.2)	/

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/: not reported in HCD.

^(a): maximum incidence.

^(b): mean incidence.

Table 14: Fetal soft tissue malformations

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses	24	23	23	24	387
Number of live fetuses	141	136	135	146	2404
Litters affected, n (%)	1 (4.2)	1 (4.3)	1 (4.3)	1 (4.2)	7 (1.8)^(b)
Fetuses affected, n (%)	1 (0.7)	1 (0.7)	1 (0.7)	1 (0.7)	13 (0.5)^(b)
Marked dilated renal pelvis, F(L)	0.7 (4.2)	0.7 (4.3)	0.7 (4.3)	0 (0)	2.4 (4.8)^(a)
Marked dilated ureter, F(L)	0 (0)	0.7 (4.3)	0.7 (4.3)	0 (0)	4.8 (4.8)^(a)
Absent kidney, F (L)	0 (0)	0 (0)	0 (0)	0.7 (4.2)	0.7 (4.5)^(a)
Absent ureter, F(L)	0 (0)	0 (0)	0 (0)	0.7 (4.2)	0 (0.0)

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

^(a): maximum incidence.

^(b): mean incidence.

Conclusions:

The test item was administered to pregnant Sprague-Dawley rats by gavage, once daily, from Day 6 to Day 20 p.c., inclusive, at the dose levels of 100, 300 and 1000 mg/kg/day. The control group received the vehicle, PEG 400, under the same experimental conditions.

On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day based on ptyalism,
- the No Observed Effect Level (NOEL) for embryo-fetal development was considered to be 1000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation until the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.) inclusive].

This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001).

 

Methods

Three groups of 24 time-mated female Sprague-Dawley rats received the test item by the oral route (gavage), at a dose level of 100, 300 or 1000 mg/kg/day, once daily from Day 6 to Day 20 p.c. inclusive. Another group of 24 time-mated female rats received the vehicle only, PEG 400, under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The test item concentrations in the dose formulations were determined. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 21 p.c., the females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage). Macroscopic lesions were collected from all dams and preserved in 10% buffered formalin.

 

Results

Chemical analyses

The concentrations of the test item in the dose formulations performed on Weeks 1 and 3 (-1.8% to +8.6% of the nominal concentrations) remained within an acceptable range of variations (± 15% of the nominal concentrations).

No test item was observed in the control dose formulation.

 

Pregnancy status

All females were pregnant with the exception of two (one in the 100 and one in the 300 mg/kg/day groups).

 

Mortality

There were no unscheduled deaths.

 

Clinical signs

Ptyalism was noted in 13/24 females given 100 mg/kg/day, 22/24 females given 300 mg/kg/day and 21/24 females given 1000 mg/kg/day. This finding was considered to be test item-related but not adverse.

 

Body weight, body weight change and food consumption

There were no effects.

 

Macroscopic post-mortem examination

No test item-related findings were observed.

 

Net body weight change and hysterectomy data

No test item-related changes were observed in the gravid uterus weight, carcass weight, net body weight change or hysterectomy data.

 

Fetal body weight and sex-ratio

There were no effects on fetal body weight or sex-ratio.

 

Fetal examinations

External examination:

. there were no test item-related variations or malformations at external examination.

 

Soft tissue examination:

. there were no test item-related variations or malformations at soft tissue examination.

 

Cartilage and skeletal examinations:

. there were no relevant differences from controls in the frequency of skeletal variations and no test item-related malformations at skeletal examination.

 

Conclusion

The test item was administered to pregnant Sprague-Dawley rats by gavage, once daily, from Day 6 to Day 20 p.c., inclusive, at the dose levels of 100, 300 and 1000 mg/kg/day. The control group received the vehicle, PEG 400, under the same experimental conditions.

 

On the basis of the results obtained in this study:

. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day based on ptyalism,

. the No Observed Effect Level (NOEL) for embryo-fetal development was considered to be 1000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is considered to be reliable with a klimisch score of 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The objective of this study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation until the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.) inclusive]. This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001).

 

Three groups of 24 time-mated female Sprague-Dawley rats received the test item by the oral route (gavage), at a dose level of 100, 300 or 1000 mg/kg/day, once daily from Day 6 to Day 20 p.c.inclusive. Another group of 24 time-mated female rats received the vehicle only, PEG 400, under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The test item concentrations in the dose formulations were determined. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 21p.c., the females were euthanized and a macroscopicpost-mortemexamination was performed. Hysterectomy was performed and the numbers ofcorpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage). Macroscopic lesions were collected from all dams and preserved in 10% buffered formalin.

 

All females were pregnant with the exception of two (one in the 100 and one in the 300 mg/kg/day groups).

There were no unscheduled deaths. Ptyalism was noted in 13/24 females given 100 mg/kg/day, 22/24 females given 300 mg/kg/day and 21/24 females given 1000 mg/kg/day. This finding was considered to be test item-related but not adverse. There were no effects on Body weight, body weight change and food consumption.

No test item-related macroscopic post-mortem findings were observed.

No test item-related changes were observed in the gravid uterus weight, carcass weight, net body weight change or hysterectomy data.

There were no effects on fetal body weight or sex-ratio.

There were no test item-related variations or malformations at external examination or at soft tissue examination.

There were no relevant differences from controls in the frequency of skeletal variations and no test item-related malformations at skeletal examination.

  

On the basis of the results obtained in this study:

. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day based on ptyalism,

. the No Observed Effect Level (NOEL) for embryo-fetal development was considered to be 1000 mg/kg/day.

Justification for classification or non-classification

Based on the available data, no classification for reproduction is required for 2 moles ethoxylated bisphenol A dimethacrylate according to the Regulation EC n°1272/2008.

Justification: Absence of adverse effects on fertility, reproduction parameters and on foetal development in the available studies performed on the registered substance.