Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In a recent GLP combined repeated dose and reproduction / developmental screening test conducted according to OECD guideline 422, the NOAEL for the reproductive/developmental toxicity was concluded to be higher than 900 mg/kg bw/day in this study.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 February 2010 to 1 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 301 to 348 g for males and 209 to 240 g for females
- Housing: up to 5 during pre-mating for all animals and after mating for males, individually with litter for females during gestation and littering.
- Diet (e.g. ad libitum): standard rodent diet (SDS VRF1 Certified) ad libitum, except overnight before routine blood sampling for Toxicity subgroup animals.
- Water (e.g. ad libitum): potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 February 2010 To: 12 April 2010
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: approximately 50% of the final volume of corn oil was added to the required amount of test material. The formulation was mixed using a magnetic stirrer until all of the test material had dissolved and more corn oil was added to make up the required volume. The formulation was then mixed using a magnetic stirrer until homogeneous.
Initially all formulations were prepared freshly on the day of use and used within two hours of completion of preparation. However, following confirmation of the results from a homogeneity and stability study (Huntingdon Life Sciences Study No. RAJ0008) formulations were prepared freshly each week and stored refrigerated (approximately 4°C) until required for use.

VEHICLE
- Concentration in vehicle: 20, 60 and 180 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5 mL/kg bw/day
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in the first and last weeks of the dosing period were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. Two assays from each group were analysed. The mean concentrations of CAS 3290-92-4 in test formulations analysed for the study were within applied limits, +10%/-15% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
Five consecutive weeks for Toxicity subgroup males and females and Reproductive subgroup males. Reproductive subgroup females were treated daily for two weeks prior to pairing, throughout pairing and up until the day prior to termination on Day 7 of lactation. Offspring were not dosed.
Frequency of treatment:
Once a day, 7 days a week
Details on study schedule:
None
Remarks:
Doses / Concentrations:
100, 300 and 900 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Toxicity subgroup: 5 males and 5 females/dose
Reproductive subgroup: 5 males and 10 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in a preliminary study (Huntingdon Life Sciences Study Number: RAJ0003), dose levels of 80, 250, 750 and 1000 mg/kg bw/day were employed and there was a suggestion of reduced bodyweight gain among males and females receiving 1000 mg/kg bw/day, and liver and kidney weights were high in all groups. There was no evidence of an effect of treatment on clinical condition, food consumption or oestrous cycles at any dose level investigated, and no treatment-related macroscopic abnormalities were observed. For the current study, treatment would continue for approximately 5 weeks and females would be pregnant, therefore, in recognition of the potential for reduced bodyweight gain, the high dose level was set at 900 mg/kg bw/day, the low dose of 100 mg/kg bw/day was chosen as an anticipated No Observed Adverse Effect Level, and the intermediate dose level of 300 mg/kg bw/day at a logarithmic interval between the low and high doses.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupant(s). During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS:
Detailed observations were recorded in relation to dose administration. For the Toxicity subgroup animals and for Reproductive subgroup males these were recorded daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and on one occasion during Week 5. For Reproductive subgroup females these were recorded daily during the first week of treatment, twice weekly during Week 2 of treatment, on Days 0, 4, 7, 11, 14 and 20 after mating and Day 4 of lactation.

Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal (physical condition and behaviour during handling with particular attention to possible signs of neurotoxicity). For the Reproductive subgroup females during the post-mating period, these observations were conducted on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation.

BODY WEIGHT:
Toxicity subgroup males and females and Reproductive subgroup males were weighed weekly throughout the study. Reproductive subgroup females were weighed weekly until mating was detected, on Days 0, 7, 14 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded weekly for all animals for the first two weeks of treatment. Food consumption continued to be recorded weekly for the remaining three weeks of treatment for Toxicity subgroup females. Food consumption was not recorded during Week 3 of treatment for Toxicity subgroup males or Reproductive subgroup males as the animals were in pairing with the Reproductive subgroup females during this period. Food consumption was recorded, however, during Weeks 4 and 5 of treatment for these males.
For each Reproductive subgroup female, the weight of food supplied, that remaining and an estimate of any spilled was recorded for the periods Days 0-6, 7-13 and 14-19 after mating and Days 1-3 and 4-6 of lactation.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation.

OTHER:
- SENSORY REACTIVITY:
Sensory reactivity and grip strength assessments were performed (before dosing) on all Toxicity subgroup animals during Week 5 of treatment. The following measurements, reflexes and responses were recorded: approach response, touch response, auditory startle reflex, tail pinch response and grip strength.

- MOTOR ACTIVITY:
During Week 5 of treatment (before dosing), the motor activity of each Toxicity subgroup animal was measured using a Rodent Activity Monitoring System (Version 2.0.3). Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total).

- HAEMATOLOGY:
During Week 5 of treatment (after sensory reactivity, grip strength and motor activity assessments), blood samples were obtained from all Toxicity subgroup animals after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein. The following were measured using a Bayer Advia 120 haematology analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total leucocyte count (WBC), Differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)) and Platelet count (Plt). The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded. Prothrombin time (PT) (using an ACL 3000 Plus analyser and IL PT-Fibrinogen reagent) and Activated partial thromboplastin time (APTT) (using an ACL 3000 Plus Analyser and IL APTT reagent) were also measured.

- BLOOD CHEMISTRY:
At the same time and using the same animals as for peripheral haematology, the plasma was examined using a Roche P Modular Analyser for: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile Acids (BIAC), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb) (by chemical assay).
Oestrous cyclicity (parental animals):
For 15 days before pairing (including the day of pairing), daily vaginal smears (dry) were taken from all Reproductive subgroup females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.
Sperm parameters (parental animals):
No data
Litter observations:
PARAMETERS EXAMINED:
All litters were examined at approximately 24 hours after birth and then daily thereafter for clinical signs (evidence of ill health or reaction to treatment), litter size (mortality and consequent changes in litter size from Days 1-7 of age), sex ratio of each litter (recorded on Days 1, 4 and 7 of age) and individual bodyweight (recorded on Days 1, 4 and 7 of age).

GROSS EXAMINATION OF PUPS:
All live pups were sacrificed on day 7 and were subject to a detailed necropsy. Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were examined.
Postmortem examinations (parental animals):
SACRIFICE:
Toxicity subgroup males and females and Reproductive subgroup males were killed in Week 6 after completion of the Week 5 investigations.
Reproductive subgroup females were killed on Day 7 of lactation.

GROSS NECROPSY:
All animals were subject to a detailed necropsy. For Reproductive subgroup females, the number of uterine implantation sites was also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues indicated in Table 1 were weighed.
In the Toxicity subgroup, the following organs were fixed for histopathology: Adrenal glands, Peyer’s patch, Brain, Pituitary, Caecum, Prostate, Colon, Rectum, Duodenum, Sciatic nerves, Epididymides (L&R), Seminal vesicles and coagulation gland, Heart, Spinal cord, Ileum, Spleen, Jejunum, Sternum with marrow, Kidneys, Stomach, Liver, Testes (L&R), Lungs, Thymus, Lymph nodes (left axillary and mesenteric), Thyroid with parathyroids, Trachea, Mammary area (caudal), Urinary bladder, Oesophagus, Uterus with cervix and oviducts, Ovaries (L&R) and Vagina.
In the Reproductive subgroup, the following organs were fixed for histopathology: Mammary area (caudal), Testes (L&R), Ovaries (L&R), Uterus with cervix and oviducts, Pituitary, Vagina and Prostate. Samples of any abnormal tissues were also retained and processed for examination.
All tissues preserved for examination were examined for all Toxicity subgroup and Reproductive subgroup animals of Control group and 900 mg/kg bw/day group. Tissues reported at macroscopic examination as being grossly abnormal were examined for all animals.
Postmortem examinations (offspring):
All live pups were sacrificed on day 7 and were subject to a detailed necropsy. Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were also examined.
Statistics:
The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, food consumption, organ weight, litter size and survival indices and clinical pathology data: 1) a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. 2) a non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.
For organ weight data, analysis of covariance was performed. Sex ratio were analysed by Wald chi-square test.
For gestation length an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups.
Reproductive indices:
Percentage mating : Number animals mating / Animals paired × 100
Conception rate (%) : Number animals achieving pregnancy / Animals mated × 100
Fertility index (%) : Number animals achieving pregnancy / Animals pairing × 100
Offspring viability indices:
Gestation index (%) : Number of live litters born / Number pregnant × 100
Post - implantation survival index (%) : Total number offspring born / Total number uterine implantation sites × 100
Live birth index (%) : Number live offspring on Day 1 after littering / Total number of offspring born × 100
Viability index (%) : Number live offspring on Day 4 after littering / Number live offspring on Day 1 after littering × 100
Lactation index (%) : Number live offspring on Day 7 after littering / Number live offspring on Day 1 after littering × 100
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One Reproductive subgroup female receiving 100 mg/kg/day was killed for welfare reasons on Day 22 of gestation due to dystocia. This female had shown evidence of parturition for approximately 4 hours in the absence of any pups being born, and showed signs of underactive behaviour, pallor, piloerection, fast respiration and blood discharge from the vagina immediately prior to despatch to necropsy. Macroscopic examination revealed twelve live and three dead fetuses in utero, and dark contents in the lower gastro-intestinal tract. In the absence of similar instances of dystocia, the demise of this female in the lowest dose group was considered incidental and unrelated to treatment.
One Reproductive subgroup Control female was killed for welfare reasons prior to dose administration on Day 4 of lactation; as a consequence the litter was also sacrificed. The female showed signs of underactive behaviour, pallor, dark eyes and gasping respiration immediately prior to despatch to necropsy. Macroscopic examination revealed clear fluid in the thoracic cavity with pale adhesions attached to all surfaces, dark areas on the lungs and trauma to the trachea; these findings would suggest that this animal had been mis-dosed at some point in the previous few days.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Among males receiving 900 mg/kg bw/day, mean bodyweight gain was slightly lower than Control during the two week pre-pairing period, with the difference in weight gain between Week 1 and Week 2 of treatment attaining statistical significance. Thereafter, mean weight gain was essentially similar to Control and overall gain during the treatment period (Week 0 to Week 5) was 92% of Control. The mean bodyweight gain of males receiving 300 or 100 mg/kg bw/day was considered unaffected by treatment.
Among females receiving 900 mg/kg bw/day, mean bodyweight gain was statistically significantly lower than Control during the first week of treatment. Thereafter, mean weight gain was similar or superior to Control, such that overall gain during the treatment period for the Toxicity subgroup females was 114% of Control.
For the Reproductive subgroup females at 900 mg/kg bw/day, mean bodyweight at pairing was slightly low, setting the baseline at the start of gestation. There was no effect of treatment on the mean bodyweight gain of Reproductive subgroup females during gestation or lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food intake of males and females in all dose groups was considered unaffected by treatment throughout the study.
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):
Assessment of haematological parameters for Toxicity phase animals during Week 5 of treatment revealed some minor inter-group differences but no clear evidence of an adverse effect of treatment.
Among females receiving 900 mg/kg bw/day, mean cell haemoglobin concentration was slightly lower than Control and monocyte concentrations were slightly higher than Control, with differences attaining statistical significance. In the absence of any other differences in the erythrocyte or leucocyte parameters, these minor differences were considered to be of no toxicological importance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
During Week 5, slightly high plasma potassium and albumin concentrations were apparent in Toxicity subgroup males receiving 900 mg/kg bw/day, slightly high phosphorous plasma concentration in males receiving 300 or 900 mg/kg bw/day and slightly low sodium among females at 900 mg/kg bw/day. In view of the differences in response between the sexes, the toxicological significance of these minor differences is uncertain.
All other inter-group differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
SENSORY REACTIVITY OBSERVATIONS AND GRIP STRENGTH: Sensory reactivity observations and grip strength values for Toxicity subgroup animals were similar to those for Controls, and considered unaffected by treatment.

MOTOR ACTIVITY: Motor activity scores for Toxicity subgroup males and females were considered to be unaffected by treatment.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The microscopic examination performed after five weeks of treatment revealed no test substance-related lesions. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted.
The incidence and distribution of all findings were consistent with the commonly seen background of microscopic changes.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Oestrous cycle length, pre-coital interval and mating performance and fertility of the Reproductive subgroup females was unaffected by treatment at all dose levels investigated. All animals mated at the first oestrus opportunity after cohabitation. There was a suggestion of a minor shift towards a slightly longer gestation length among Reproductive subgroup females receiving 900 mg/kg bw/day, with no females having a 22-day gestation length compared with 3, 5 and 4 females at 0, 100 and 300 mg/kg bw/day, respectively. The incidence of females with a 23.5 day gestation length was marginally outside the Historical control data range. The gestation index was 100% in all groups, with the exception of the 100 mg/kg bw/day group, where one female was killed for welfare reasons on Day 22 of gestation due to dystocia.
Dose descriptor:
NOAEL
Effect level:
> 900 mg/kg bw/day (nominal)
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:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
LITTER SIZE, SEX RATIO AND SURVIVAL INDICES: The mean number of implantation sites, and consequently mean litter size, was lower than Control among Reproductive subgroup females receiving 900 mg/kg bw/day, with 4/10 females having less than 14 implantations compared with 0/10 in the concurrent Control group; mean values were slightly outside the Historical control data range. This statistically significant reduction in the mean number of implantation sites is associated with low litter size were observed at the high dose (900 mg/kg bw/d). In this screening study, the number of corpora lutea present for each animal was not determined. Thus, it cannot be assessed whether this is a spontaneous finding related to a reduction in the number of eggs available for fertilization or indicative of a treatment-related pre-implantation loss.

There was no effect on pre- or post-natal survival at any dose level, and the percentage of males in the litters in all groups was consistent between Days 1 and 7 of age, indicating that there was no preferential mortality of either sex.

CLINICAL SIGNS (OFFSPRING): There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment.

BODY WEIGHT (OFFSPRING): At 900 mg/kg bw/day, mean male and female offspring bodyweights on Day 1 of age were higher than Control, with statistical significance achieved for females; mean absolute bodyweight remained higher than Control to Day 7 of age. Mean bodyweight gain of males and females from birth to Day 4 of age was statistically higher than concurrent Control. These differences were attributed to the slightly lower litter size and slight shift in gestation length observed in this group.
Offspring bodyweight and bodyweight gain were unaffected by maternal treatment at 100 and 300 mg/kg bw/day.

GROSS PATHOLOGY (OFFSPRING): The type and distribution of findings at necropsy of the offspring that survived to scheduled termination on Day 7 of age did not suggest any adverse effect of treatment of the Reproductive subgroup females.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 900 mg/kg bw/day (nominal)
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:
not specified
Conclusions:
Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity in the CD rat following 5 weeks of treatment was higher than 900 mg/kg bw/day. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be higher than 900 mg/kg bw/day.
Executive summary:

In a GLP study conducted according to OECD guideline 422, three groups each comprising five male and five female rats for the Toxicity subgroup and five male and ten female rats for the Reproductive subgroup received propylidynetrimethyl trimethacrylate at doses of 100, 300 or 900 mg/kg bw/day. Toxicity subgroup males and females and Reproductive subgroup males were treated daily for five consecutive weeks. Reproductive subgroup females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 7 of lactation. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume-dose.

During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

There were two mortalities during the course of the study. One Reproductive subgroup female receiving 100 mg/kg bw/day was killed for welfare reasons on Day 22 of gestation due to dystocia. In addition, one Reproductive subgroup Control female (and consequently the litter) was killed for welfare reasons prior to dose administration on Day 4 of lactation. These isolated incidents were considered unrelated to treatment.

There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. There was also no effect of treatment on the food consumption, sensory reactivity findings, grip strength values and motor activity in males and females throughout the study.

At 900 mg/kg bw/day, mean bodyweight gain of males was slightly lower than Control during the two week pre-pairing period; females in this dose group showed similarly low weight gain during the first week of treatment only. Thereafter, for both sexes mean weight gain was essentially similar to Control, and the bodyweight gain of females during gestation and lactation was unaffected by treatment.

Oestrous cycle length, pre-coital interval and mating performance and fertility of the Reproductive subgroup females was unaffected by treatment. There was a suggestion of a minor shift towards a slightly longer gestation length among Reproductive subgroup females receiving 900 mg/kg bw/day.

Among the Toxicity subgroup animals, liver weights were slightly high in males and females receiving 900 mg/kg bw/day, and kidney weights were also high among females in this dose group. There were no associated haematological or biochemical changes, or macroscopic/microscopic abnormalities to explain the difference in the weight of the organs.

There were no macroscopic abnormalities and no test substance-related lesions at microscopic examination.

There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment.

A statistically significant reduction in the mean number of implantation sites, associated with low litter size were observed at the high dose (900 mg/kg bw/d). In this screening study, the number of corpora lutea present for each animal was not determined. Thus, it cannot be assessed whether this is a spontaneous finding related to a reduction in the number of eggs available for fertilization or indicative of a treatment-related pre-implantation loss.

There was no effect on pre- or post-natal survival and on sex ratio at any dose level. At 900 mg/kg bw/day, mean male and female offspring bodyweights on Day 1 of age were higher than Control; these differences were attributed to the slightly lower litter size and slight shift in gestation length observed in this group. There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment.

Based on the results of this study, it was concluded that the NOAEL for systemic toxicity in the CD rat following 5 weeks of treatment was higher than 900 mg/kg bw/day. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be higher than 900 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
900 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Stannard's study is considered to be reliable, and was performed according to the OECD 422 guideline.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Oral combined repeated dose and reproduction / developmental screening test

In a GLP study conducted according to OECD guideline 422, three groups each comprising five male and five female rats for the Toxicity subgroup and five male and ten female rats for the Reproductive subgroup received propylidynetrimethyl trimethacrylate at doses of 100, 300 or 900 mg/kg bw/day. Toxicity subgroup males and females and Reproductive subgroup males were treated daily for five consecutive weeks. Reproductive subgroup females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 7 of lactation. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume-dose.

There were two mortalities during the course of the study. One Reproductive subgroup female receiving 100 mg/kg bw/day was killed for welfare reasons on Day 22 of gestation due to dystocia. In addition, one Reproductive subgroup Control female (and consequently the litter) was killed for welfare reasons prior to dose administration on Day 4 of lactation. These isolated incidents were considered unrelated to treatment.

There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. There was also no effect of treatment on the food consumption, sensory reactivity findings, grip strength values and motor activity in males and females throughout the study.

At 900 mg/kg bw/day, mean bodyweight gain of males was slightly lower than Control during the two week pre-pairing period; females in this dose group showed similarly low weight gain during the first week of treatment only. Thereafter, for both sexes mean weight gain was essentially similar to Control, and the bodyweight gain of females during gestation and lactation was unaffected by treatment.

Oestrous cycle length, pre-coital interval and mating performance and fertility of the Reproductive subgroup females was unaffected by treatment. There was a suggestion of a minor shift towards a slightly longer gestation length among Reproductive subgroup females receiving 900 mg/kg bw/day.

Among the Toxicity subgroup animals, liver weights were slightly high in males and females receiving 900 mg/kg bw/day, and kidney weights were also high among females in this dose group. There were no associated haematological or biochemical changes, or macroscopic/microscopic abnormalities to explain the difference in the weight of the organs.

There were no macroscopic abnormalities and no test substance-related lesions at microscopic examination.

There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment.

A statistically significant reduction in the mean number of implantation sites, associated with low litter size were observed at the high dose (900 mg/kg bw/d). In this screening study, the number of corpora lutea present for each animal was not determined. Thus, it cannot be assessed whether this is a spontaneous finding related to a reduction in the number of eggs available for fertilization or indicative of a treatment-related pre-implantation loss.

There was no effect on pre- or post-natal survival and on sex ratio at any dose level. At 900 mg/kg bw/day, mean male and female offspring bodyweights on Day 1 of age were higher than Control; these differences were attributed to the slightly lower litter size and slight shift in gestation length observed in this group. There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment.

Based on the results of this study, it was concluded that the NOAEL for systemic toxicity in the CD rat following 5 weeks of treatment was higher than 900 mg/kg bw/day. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be higher than 900 mg/kg bw/day.


Effects on developmental toxicity

Description of key information
Developmental toxicity of TMPTMA was studied in rat and rabbit species. 
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 02-Sep-2014 to 18-Mar-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22nd January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
EC 2004/73 B31, dated April 29, 2004
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
EPA 712-C-98-207, dated August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000).
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks at day 0 post coitum
- Weight at study initiation: 178 to 281 g at day 0 post coitum
- Fasting period before study: None
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK and ISO-BLOX from Harlan Laboratories B.V. / Netherlands).
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 13/14) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed was analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of respective samples were performed.
- Acclimation period: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 02-Sep-2014 (start of 7-day Acclimatization) To: 09-Oct-2014 (Last Necropsy)
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
The vehicle was pre-warmed to a temperature of approximately 40 °C. Propylidynetrimethyl trimethacrylate was weighed into a glass beaker on a tared precision balance and approximately 80 % of the warm vehicle was added (w/v). The mixture was homogenized using an electrical homogenizer until a clear to colloid-like yellowish solution was obtained. Having obtained a homogeneous mixture, the remaining vehicle was added until the required final volume. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Stability and Storage of Dose Formulations: For at least 8 days at room temperature (20 ± 5 °C) based on stability results of Harlan Laboratories Study no. D84010 (Braun 2015, subchronic repeated oral toxicity

DIET PREPARATION
- Not relevant due to exposure via gavage

VEHICLE
- Justification for use and choice of vehicle: Considered non-toxic to the test animals and ability to form a homogeneous mixture with the test item
- Concentration in vehicle (nominal): 0, 25, 75 and 250 mg/mL (at 0, 100, 300 and 1000 mg/kg bw/day, respectively)
- Amount of vehicle (by gavage): 4 mL/kg body weight with a daily adjustment to the actual body weight
- Lot/batch no.: 453206485 (Source: Carl Roth GmbH + Co. KG, Karlsruhe/Germany, Expiry Date: 14-Jan-2016)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples were dispatched to the analytical laboratories internally (at room temperature) and stored frozen at -20 ± 5 °C until analysis.
The test item was used as the analytical standard.
Stock solutions of Propylidynetrimethyl trimethacrylate in acetonitrile were prepared for external calibration. For example, 34.77 mg of Propylidynetrimethyl trimethacrylate was weighed into a 50 mL volumetric flask and filled to about 75% of final volume with acetonitrile. The mixture was sonicated for at least five minutes and brought to volume with acetonitrile to yield a solution with a concentration of 695.4 µg/mL. Aliquots of this stock calibration solution were diluted with acetonitrile to obtain calibration solutions with nominal concentrations ranging from 10.43 to 99.44 µg/mL. On each occasion calibration solutions derived from two stock solutions were used for calibration.
Each sample was transferred into an appropriate volumetric flask. The sample vial was successively rinsed with at least two portions of tetrahydrofuran and the rinsings were combined in the volumetric flask. The flask was filled to about 75 % of the target volume with tetrahydrofuran and dissolution was achieved by sonication for at least five minutes. The flask was filled to the mark with tetrahydrofuran. Sample solutions were further diluted with acetonitrile into the calibration range.
Gas Chromatographic Conditions:
- Computerized System: EZ Chrom Elite; Agilent Technologies
- Column: BGB 1701 - BGB; 30 m x 0.25 mm; 0.25 µm
- Carrier Gas: Helium
- Temperature Gradient:
Rate [°C/min]; Temp. [°C]; Time [min]
0; 200; 0
10; 300; 5
- Running Time: 15 min
- Injector Temperature: 230 °C
- Injection Mode: Split ratio 5:1
- Flow: 1 mL/min (constant flow)
- Detector: FID; 250 °C (H2: 31; Air: 380; N2: 28 mL/min)
- Injection Volume: 2 µL
- Retention Time: Ca. 4.4 min
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ±20 % variation from the calibration curve derived by linear regression analysis. The coefficients of determination (R²) were higher than 0.99.
The Propylidynetrimethyl trimethacrylate peak was assigned in sample chromatograms by comparison to that of calibration solutions. In blank sample chromatograms no peak appeared at the retention time of Propylidynetrimethyl trimethacrylate and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed.
The Propylidynetrimethyl trimethacrylate concentrations in the dose formulations ranged from 81.5 to 92.5 % with reference to the nominal and were within the accepted range of ±20 %. The homogeneous distribution of Propylidynetrimethyl trimethacrylate in the preparations was approved because single results found did not deviate more than 1.3 % from the corresponding mean and met the specified acceptance criterion of =15 %. Accordingly the effects were assigned to the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: Cohoused, After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- M/F ratio per cage: 1:1
- Length of cohabitation: After 11 days (08 to 18-Sep-2014) pairing of all test animals was successfully completed.
- Further matings: No
- Verification of same strain and source of both sexes: Yes, all animals belonged to the RccHan™: WIST(SPF)
- Proof of pregnancy: Vaginal plug observed or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: None
Duration of treatment / exposure:
15 days (during gestation period from day 6 to 20 post coitum)
Frequency of treatment:
Daily, at approximately 24 hour intervals
Duration of test:
21 days post coitum
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
24 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range-finding study in Han Wistar rats, Harlan Laboratories non-GLP study no. D84021, using dose levels of 0, 100, 300 and 1000 mg/kg/day.
- Rationale for animal assignment: Computer-generated random algorithm
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Viability / Mortality observation were made twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
- Time schedule: For the following periods: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post coitum
- Organs examined: Gross macroscopic examination of all internal organs
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Post mortem examination with emphasis on the uterus, uterine contents, corpora lutea count and position of foetuses in the uterus was performed and the data recorded.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions (embryonic): Yes
- Number of late resorptions (foetal): Yes
- Other: The uteri (and contents) of all females with live foetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Foetuses were removed from the uterus by caesarean section, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
1. Microdissection technique (sectioning/dissection technique): Approximately one half of the foetuses from each litter were fixed in Bouin's fixative. They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
2. The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol, carcasses were processed through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The assessment included, but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.

The foetuses were sent to the contributing scientist for peer review at Huntingdon Life Sciences.
Statistics:
The following statistical methods were used to analyse food consumption, body weights, reproduction and skeletal examination:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied, if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied, if the variables could be dichotomized without loss of information.
Indices:
No indices were calculated as percentages were used.
Historical control data:
Tables summarizing the available historical control data from six studies performed at the test site (referenced 12/02, 12/03, 12/04, 13/01, 13/02 and 13/03) are given in Appendix V of the study report.
Historical control data were used in the discussion of foetal Visceral Abnormalities and Variations (incidence of the left-sided umbilical artery in group 4 marginally exceeded the upper range of the historical control data).
The number of foetal retinal folds observed in the control group was not in accordance with the occurrence reported in the historical control values where no retinal folds were observed. This could be explained by the fact that the source of the Bouin’s fixative for the present study was different from that used in studies that compiled the historical control data; this difference was considered to be a potential cause for the slight (but not clearly dose-related) increase in retinal folds noted (see Discussion of Retinal Folds observations, below).
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical symptoms or signs were observed at any dose level during the gestation period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females survived until the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effects on mean absolute body weights were recorded.
The mean body weight gain was significantly lower (p<0.01) from day 9 p.c. in group 4 when compared to the controls (40 % in group 4 vs. 46 % in the controls on day 21).
The corrected body weight gain (corrected for the gravid uterus weight) was significant lower in group 4 (p<0.01) when compared to the controls (8.0 vs. 11.5 % in the controls on day 21). The effects on body weight gain and corrected body weight gain in group 4 were considered test item-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the food consumption during the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings were observed at any dose level during macroscopic examination.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
All females survived until the scheduled necropsy. No clinical signs were recorded in any group and the mean daily food consumption of all groups compared favourably. Although the mean absolute body weights were unaffected, a statistically significant lower mean body weight gain in group 4 (40 vs. 46 % in the controls on day 21 post coitum) and a statistically significant decreased corrected body weight gain (corrected for the gravid uterus weight) in group 4 (8.0 vs. 11.5 % in the controls on day 21) were considered to be test item-related. The reproduction data (post-implantation loss and mean number of foetuses per dam) was unaffected by treatment and no macroscopical findings were noted at any dose level.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Placenta weights were not affected by the treatment with the test item at any dose level. Mean placenta weights calculated on a litter basis were: 0.505 g, 0.485 g, 0.490 g and 0.495 g whereas calculated on an individual basis, they were 0.503 g, 0.483 g, 0.489 g and 0.495 g, both cited in order of ascending dose level. Mean values calculated on an individual basis were significantly lower in group 2 (p<0.05) when compared to the control. In the absence of a dose-response relationship, the difference was considered to be unrelated to the treatment.
Details on maternal toxic effects:
The statistically significant decrease in the implantation sites and increase in pre-implantation loss in group 2 was not test item-related since this was a single outlying value (female no. 48) and occurred before treatment started.
The relevant reproduction data (post implantation loss and number of foetuses per dam) were not affected by treatment with the test item.
Dose descriptor:
NOEL
Remarks:
(maternal toxicity)
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Remarks:
(maternal toxicity)
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
effects observed, treatment-related
Fetal body weight changes:
no effects observed
Description (incidence and severity):
When compared with the controls, lower mean body weights of live foetuses were noted in group 4 (-6.1 % on litter basis and -8.2 % on individual basis). The difference attained statistical significance (p<0.01) and was considered to be test item-related. Both male and female foetuses in group 4 were significantly lower (all p<0.01) when compared with the controls.
The mean live foetus body weight of group 3 was similar to that of the control group.
The individual and litter mean body weight of live foetuses in group 2 was marginally lower (-2 %) than the controls; the reduction noted in mean individual foetus weights was statistically significant but unrelated to dose and therefore considered to be unrelated to the test item.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects on the sex ratio of the foetuses were noted in any group.
The proportion of male foetuses was 49.4, 54.7, 49.8 and 50.4 % in order of ascending dose level.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related findings were observed during external examination of the foetuses.
Umbilical hernia was recorded in one female foetus of group 3. A domed head, protruding tongue, and short and flexed forelimbs were recorded in one male foetus of group 4. These findings were recorded in single animals and therefore considered to be incidental.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Bone and Cartilage Abnormalities and Variations
During skeletal examination of the foetuses, findings were noted in:
15 % examined foetuses (in 67 % litters) in group 1
15 % examined foetuses (in 57 % litters) in group 2
17 % examined foetuses (in 67 % litters) in group 3
17 % examined foetuses (in 60 % litters) in group 4
A small number of bone and cartilage abnormalities were noted in treated and control groups. The incidence of the findings in the treated groups was low, were not related to dose and considered to be of no toxicological relevance.
All bone and cartilage variations noted in treated groups were considered to be unrelated to the test item. The incidence of the findings in the treated groups was low and unrelated to dose.

Ossification and Supernumerary Ribs
There were no test item-related effects on the stage of development in any dose group.
The significantly increased incidence of incomplete ossification of sternabrae 5 or non-ossification of calcanei (bilateral) were considered likely to be a secondary result of the lower mean dam body weight gain and/or foetal body weights. These structures typically show high rates of non- or incomplete ossification in control rat foetuses and therefore a relationship with the test item treatment is considered unlikely.

Additional Cartilage Variations
All additional cartilage variations noted in treated groups were considered to be unrelated to the test item. The incidence of the findings in the treated groups was low and unrelated to dose.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During visceral examination of the foetuses, findings were noted in:
63 % examined foetuses (in 100 % litters) in group 1
62 % examined foetuses (in 100 % litters) in group 2
57 % examined foetuses (in 100 % litters) in group 3
68 % examined foetuses (in 100 % litters) in group 4
No test item-related visceral abnormalities were noted. All abnormal changes were noted at low incidence and were unrelated to dose.
The high incidence of retinal folds noted in the foetuses of groups 3 and 4 was considered to be an artefact of fixation. All other changes were noted at low incidence and were unrelated to dose.
Although the incidence of the left-sided umbilical artery in group 4 marginally exceeded the upper range of the historical control data, the finding was noted at lower incidence in group 3 than in either group 2 or the control group. Therefore, this latter difference was considered to be of no toxicological relevance.
Details on embryotoxic / teratogenic effects:
The mean placenta weights of all groups were similar. The external examination of the foetuses showed no abnormalities of toxicological relevance and sex ratios were unaffected. Foetuses of group 4 dams had lower mean body weights (-6 % on litter basis and -8 % on individual basis). This was considered to be related to the treatment with test item.

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Summary of Performance of Mated Females

Group
Dose (mg/kg)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

1 - 24

25 - 48

49 - 72

73 - 96

Number of mated females

24

24

24

24

Not pregnant (A)

3

3

3

4

Number of females with live foetuses at termination*

21

21

21

20

* Only dams with at least one live foetus at caesarean section were used for the calculations of food consumption, body weight gain and corrected body weight gain data.

(A) Females no. 4, 15, 24, 35, 43, 47, 49, 56, 61, 73, 78, 83, and 87 were not pregnant.

Conclusions:
NOEL maternal toxicity 300 mg/kg bw/day, NOAEL maternal toxicity 1000 mg/kg bw/day or higher (body weight effects)
NOEL and the NOAEL prenatal development 300 mg/kg/day (lower mean foetal weights).
Executive summary:

The oral (gavage) prenatal developmental toxicity of the test item Propylidynetrimethyl trimethacrylate (CAS 3290-92-4) to Wistar rats was investigated in a GLP-compliant dose-effect study according to the OECD TG 414 (2001) guideline.

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to caesarean section). Four groups of 24 mated females per group were treated by gavage once daily at nominal dose levels of 0 (control group), 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively). A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil). All females were sacrificed on day 21 post coitum and the foetuses were removed by caesarean section.

All females survived until the scheduled necropsy. No clinical signs were recorded in any group, and the mean daily food consumption of all groups compared favourably. Although the mean absolute body weights were unaffected, a statistically significant lower mean body weight gain of the dams in group 4 (40 vs. 46 % in the controls on day 21 post coitum) and a statistically significant decreased corrected body weight gain (corrected for the gravid uterus weight) in group 4 (8.0 vs. 11.5 % in the controls on day 21) were considered to be test item-related. The reproduction data (post-implantation loss and mean number of foetuses per dam) was unaffected by treatment and no macroscopical findings were noted at any dose level.

The mean placenta weights of all groups were similar. The external examination of the foetuses showed no abnormalities of toxicological relevance and sex ratios were unaffected. Foetuses of group 4 dams had lower mean body weights (-6 % on litter basis and -8 % on individual basis). This was considered to be related to the treatment with test item.

In conclusion based on the slightly lower mean body weight gain, the NOEL (No Observed Effect Level) for maternal toxicity was considered to be 300 mg/kg bw/day, whereas the NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be 1000 mg/kg bw/day or higher. For prenatal development, the NOEL (No Observed Effect Level) and the NOAEL (No Observed Adverse Effect Level) were considered to be 300 mg/kg/day, based on the lower mean foetal weights noted at 1000 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 May 2017 - 04 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Centre LAGO (Vonnas, France)
- Age: at the beginning of the treatment period, the animals were 18-20 weeks old
- Mean body weight: at the beginning of the treatment period, the animals had a mean body weight of 3772 g (range: 3180 g to 4580 g)
- Fasting period before study: no
- Housing: the animals were individually housed in noryl cages (Tecniplast, 4200 cm²)
- Diet: (SAFE, Augy, France) 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): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 5 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 8 h/16 h.

IN-LIFE DATES: 03 July 2017 to 04 August 2017.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% Carboxymethylcellulose (400-800 cps) / 0.5% Tween 80 in drinking water treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Justification for use and choice of vehicle: suitable formulation in the selected vehicle
- Concentration in vehicle: 33.3, 100 and 333.3 mg/mL
- Amount of vehicle: 3 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: Gas Chromatography with FID detection (GC-FID)
Test item concentrations: a sample was taken from control and test item dose formulations and analyzed using the validated method
Stability / Homogeneity: The dose formulations containing the test item and prepared at 2 mg/mL and 333.3 mg/mL in 0.5% (w/v) carboxymethylcellulose and 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis were found to be homogeneous and stable after 5 and 9 days at room temperature and protected from light.
Details on mating procedure:
- Impregnation procedure: purchased time pregnant.
- Proof of pregnancy: vaginal plug (at the breeder's facility); referred to as Day 0 post-coitum.
Duration of treatment / exposure:
The dose formulations were administered daily from Day 6 to Day 28 p.c., inclusive.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
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:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, based on the results of a preliminary study on embryo-fetal development in New Zealand White rabbits where the test item was administered by the oral route from Day 6 to Day 18 p.c. at dose levels of 100, 300 or 1000 mg/kg/day.
At 100 mg/kg/day, no signs of maternal toxicity were noted and no effects on hysterectomy data or fetuses were observed.
At 300 mg/kg/day, no signs of maternal toxicity were noted. There were no significant signs of toxicity on the number of fetuses, the number of implantation sites or on post-implantation loss. Fetal examination did not reveal any malformations. Lower fetal body weight was recorded (34.5 g vs. 39.4 g in controls; p < 0.05).
At 1000 mg/kg/day, a body weight loss was recorded between Days 6 and 9 p.c. (-32 g), associated with lower food consumption (-12% vs. controls). Lower food consumption was observed again between Days 24 and 29 p.c. (-20% vs. controls), but there were no statistical differences on the body weight or food consumption at the end of the study when compared with control group. No significant signs of toxicity were observed on the number of fetuses, the number of implantation sites or post-implantation loss, and fetal examination did not reveal any malformation. Lower fetal body weight was recorded (34.0 g vs. 39.4 g in controls; p < 0.05).

Therefore, 1000 mg/kg/day was selected as the high dose level for the present study. The low dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment: stratified procedure.
Maternal examinations:
MORBIDITY/MORTALITY:
- Time schedule: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during treatment period, including weekends and public holidays.
Two females of the group 3, showing signs of poor clinical condition or having aborted, were humanely euthanized.

CLINICAL OBSERVATIONS:
- Time schedule: from arrival, the animals were observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time of day, for the recording of clinical signs (including evidence of abortion).

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c., and before premature euthanasia.

FOOD CONSUMPTION:
- Time schedule: The quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 29 post-coitum.
- Examined: principal thoracic and abdominal organs.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early and late resorptions
- Number of dead and live fetuses
- Number of uterine scars
- Gross evaluation of placentas.
Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: all fetuses per litter
- Skeletal examinations: Yes: all fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other : fetal weight, fetal sex
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s 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, non-treatment-related
Description (incidence and severity):
See table 1.
There were no remarkable clinical observations in surviving animals.
In the 1000 mg/kg/day group, from Day 9 p.c. emaciated appearance was noted in female K31094. As this female had already lost 285 g (-8% of the initial body weight on Day 2 p.c.) and had nearly no food consumption (on Days 4 and 5 p.c.) during the pre-treatment period, a test item-related effect alone was considered to be unlikely.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths or abortions in the control, 100 or 1000 mg/kg/day groups.

In the 300 mg/kg/day group, there were two prematurely sacrificed females (K31050 and K31068):
-female K31050 was euthanized on humane grounds on Day 23 p.c. (emaciated appearance from Day 20 p.c., soft feces from Day 21 p.c. and hypoactivity together with abdominal breathing on Day 23 p.c.). This animal lost 21% of its body weight from Days 6 to 23 p.c. and nearly no food consumption was recorded on Days 15 to 19 p.c. At necropsy, this female (with 12 corpora lutea and 11 implantation sites: 4 dead fetuses and 7 live fetuses) showed edema on the pyloric region of the stomach mucosa, reddish depressed area(s) on the stomach mucosa and dilated colon with gas,
-female K31068 was euthanized due to abortion on Day 22 p.c. (blood and embryos in the bedding on Day 22 p.c.). At necropsy, this female (with 15 corpora lutea and 4 implatation sites, 1 uterine scar, 1 late resorption and 2 live fetuses) had no macroscopic post-mortem lesions.

These deaths were considered not to be test item-related as they were not dose-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 2.

When compared with controls, there were no effects on mean body weights or mean body weight changes at 100 or 300 mg/kg/day.

At 1000 mg/kg/day and when compared with controls, a slight mean body weight loss was noted over the first 3 days of treatment (-2 g vs. +27 g, not statistically significant), followed, from Day 12 p.c., by a lower mean body weight gain (statistically significant from Day 19 p.c. to Day 24 p.c.).
No statistically significant changes were observed in the final mean body weight of females. However, 4/22 females (K31078, K31093, K31094 and K31096) lost body weight during the gestation period from Day 6 p.c. to Day 29 p.c. Taking into account the amplitude of the body weight change over the whole treatment period ( 42%, p<0.05) and the body weight loss recorded in 4 females (corresponding to 18% of the size group), the decrease of body weight gain was considered to be test item-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See table 3.
When compared with controls, there were no statistical differences on mean food consumption at 100 or 300 mg/kg/day.

At 1000 mg/kg/day and when compared with controls, mean food consumption was lower over the first 3 days of treatment and from Day 12 p.c. A statistically significant change was observed from Day 24 p.c. to Day 29 p.c. (-25%, p<0.05) that was below the lower limit of the Historical Control Data. Females with body weight loss were more impacted.
These differences in mean food consumption correlated with lower mean body weight gain were therefore considered to be test item related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See table 5.
Discolored area(s) in the stomach were observed in one female from each test item-treated group. Taking into account the absence of similar findings in the control group, a test item relationship was not excluded.

The other macroscopic observations (lungs with colored foci/nodules, liquid content in the abdominal cavity, cyst on the periovarian region and/or gall bladder bilobed/reduced in size) were not attributed to the test item as they were reported with no dose-relationship and/or are findings routinely observed in pregnant female rabbits of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
See table 4.
There were no effects on mean gravid uterus weight mean carcass weight.

At 1000 mg/kg/day and when compared with controls and Reference Control Data, on Day 29 p.c. a slightly lower mean net body weight change (not statistically significant) was noted. This correlated with lower mean body weight gain.
Females with body weight loss were more affected and value of one female (K31096: -947 g) was below the lower individual limit of the Reference Control Data [-678 g; +389 g].
Based on these correlations, the lower mean net body weight change was therefore considered to be test item-related.
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
See table 15.
In the 300 mg/kg/day group, one female was euthanized due to abortion on Day 22 p.c.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
When compared with controls or Historical Control Data, there were no test item-related effects on hysterectomy data.

In controls, mean number of corpora lutea was below the lower limit of the Historical Control Data leading to higher mean numbers of corpora lutea and implantations at 300 and 1000 mg/kg/day (p<0.05 and p<0.01, respectively), when compared with controls. Although these differences were statistically significant, they were considered to be fortuitous as corpora lutea appearance occurs before the treatment period.
The higher mean number of late resorptions observed in the 300 mg/kg/day group was considered to be of no toxicological importance as this difference from controls was not dose-related.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No dams with total resorption.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
The higher mean number of late resorptions observed in the 300 mg/kg/day group was considered to be of no toxicological importance as this difference from controls was not dose-related.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
Number of dams with dead fetuses = 1 at 100 mg/kg/day only, not dose-related.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
No early deliveries.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
See table 15.
Dose descriptor:
NOAEL
Remarks:
for systemic maternal parameters
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: net body weight change
Dose descriptor:
NOAEL
Remarks:
for reproduction parameters
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:
effects observed, treatment-related
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 7.
From 100 mg/kg/day and when compared with controls, male and female fetuses had a slight to markedly lower mean body weight (-8% to -23% vs. controls). Female fetuses were more affected than male fetuses at 300 (males: -10% and females: -14% vs. controls) and 1000 mg/kg/day (males: -21% and females: -23% vs. controls), but mean fetal body weight values were within the range of the Historical Control Data.
The lower mean fetal body weight was considered to be related to the test item and adverse at 1000 mg/kg/day taking into account the amplitude of the difference.

Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See table 6.
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
At 300 and 1000 mg/kg/day and when compared with controls, a slightly, not statistically significant, higher mean percentage of male fetuses was noted (59.0% and 59.4%, respectively, vs. 50.2% in controls). These differences were considered to be related to the test item but of minor toxicological importance as they were of minor amplitude and close to the upper limit of the Historical Control Data.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, the mean litter values of fetal body weight were decreased when compared with controls and outside the HCD. These decreases at 1000 mg/kg/day were considered as adverse.
Description (incidence and severity):
n/a
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Variations
See table 8.
In the 1000 mg/kg/day group and when compared with controls, statistically significant, higher fetal (12/212 fetuses) and litter incidences (5/22 litters) of yellowish/greenish coloration of the amniotic fluid (p<0.001 and p<0.05, respectively) were observed. Nine out of twelve affected fetuses showed the lowest fetal weight of the litters. Taking into account the amplitude of the incidences (litter and fetal) of this non adverse finding, it was considered to be test item-related.

Other external variations (i.e. local edema on abdomen and paw hyperflexion) were considered to be unrelated to the test item treatment as they were not dose-related and/or were observed with a similar incidence in control and test item-treated groups.


Malformations
See table 9.
In the 1000 mg/kg/day group, three fetuses from three litters showed external malformations:
-fetus K31086-01 (fetal body weight: 23.1 g): ectrodactyly, left malpositioned digit and gastroschisis. This fetus also had skeletal malformations (i.e. left tibia misshapen, 2 absent metatarsal bones and left digits hindpaw and scoliosis),
-fetus K31083-11 (fetal body weight: 37.3 g): omphalocele,
-fetus K31094-01 (fetal body weight: 23.3 g): ombilical hernia. This fetus also had visceral malformations (i.e. marked dilated cerebral ventricle, bilateral).
These findings, mainly affecting the abdominal wall (mid-line defects: gastroschisis, omphalocele or ombilical hernia), were observed in three malformed fetuses from three different litters and two of these fetuses showed low fetal body weight.

In the 300 mg/kg/day group, there was no external malformation.
In the 100 mg/kg/day group, one malformed fetus: K31030-02 with cleft lip was present.
In the control group, there was no external malformation.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Cartilage
See table 12.
When compared with controls and/or Historical Control Data, there were higher litter and/or fetal incidences of fetuses with sternebra(e) cartilage present and/or misshapen, and metacarpal bone(s) cartilage present from 100 mg/kg/day and/or talus cartilage present at 1000 mg/kg/day. These findings were associated with a delayed ossification as described in the § Variations [i.e. unossification or incomplete ossification of hemisternebra(e), unossification or incomplete ossification of 6th sternebra(e), unossification or incomplete ossification of 1st metacarpal bone, unossification or incomplete ossification of talus] and were considered as test item treatment-related, but they are not considered to be adverse.

The other findings [i.e. cartilage of rib(s) branched, cartilage of forepaw median phalanx present, and/or cartilage of metatarsal bone absent; and cartilage of forepaw proximal phalanx present which is not reported in summary table] were not dose related, noted without a statistically significant occurrence and/or were within or close to the range of the Historical Control Data. Therefore a test item relationship was considered to be unlikely.

Variations
See table 13.
In the 1000 mg/kg/day group and when compared with controls and/or Historical Control Data, there were dose-related higher fetal and/or litter incidences of fetuses with incomplete ossification of interparietal, unossification or incomplete ossification of hemisternebra(e), unossification or incomplete ossification of 6th sternebra(e), unossification or incomplete ossification of 1st metacarpal bone, unossification or incomplete ossification of talus. These findings, associated with higher fetal and litter cartilage findings (see § Cartilage) and mainly observed with a statistically significant occurrence, were therefore considered to be test item-related but not adverse.

In the 300 mg/kg/day group and when compared with controls and Historical Control Data, there were higher fetal and/or litter incidences of fetuses with unossification or incomplete ossification of hemisternebra(e).
These dose-related findings, associated with higher fetal and litter cartilage findings (see § Cartilage), were therefore considered to be test item-related but not adverse.
There were also higher fetal and litter incidences of fetuses with incomplete ossification of interparietal,
incomplete ossification of 6th sternebra(e) and unossification of 1st metacarpal bone. A relationship to the test item could not be ruled out for these dose-related variations, although their incidences were within the range of the Historical Control Data.

In the 100 mg/kg/day group and when compared with controls, there were higher litter incidences of fetuses with unossification of 1st metacarpal bone and/or incomplete ossification of hemisternebra(e). A relationship to the test item could not be ruled out for these dose-related variations which were also associated with higher fetal and litter cartilage findings (see § Cartilage), although their incidences were within the range of the Historical Control Data.

The other variations [i.e. supernumerary 13th or 14th rib(s), knobby rib(s), incomplete ossification of forepaw median phalanx and/or unossified forepaw median phalanx; and unossified interparietal, misshapen sternebra(e), ribs ossification point on 13th thoracic vertebra(e), and/or unossified pubis which are not reported in the summary table] were not dose-related, noted without a statistically significant occurrence and/or were within or close to the range of the Historical Control Data. Therefore a test item relationship was considered to be unlikely.


Malformations
See table 14.
There was no test item-related increase in the frequency of skeletal malformations.

In the 300 and 1000 mg/kg/day group and when compared with controls, there were higher fetal and litter incidences of fetuses with split interparietal and fetuses with fused sternebra(e).
At 1000 mg/kg/day, litter (but not fetal) incidence of fused sternebra(e) was statistically significantly higher than that of the control group and higher than the maximal incidence of the Historical Control Data. Both findings are consistent with a developmental delay in ossification and could be considered as a normal step leading to definitive formation of the bone (De Sesso and Scialli, 2018). They were therefore considered as skeletal variations (not adverse) and not as malformations.
In the 1000 mg/kg/day group, multi-malformed fetus K31086-01 (fetal body weight: 23.1 g) showed scoliosis, misshapen left tibia and absence of two metatarsal bones and two digits in the left hindpaw. This fetus also had external malformations (i.e. ectrodactyly, malpositioned digit and gastroschisis).
The other malformations [i.e. split interparietal, absent interparietal, fused rib(s)] observed in the 100, 300 and/or 1000 mg/kg/day groups were of isolated occurrence (this could include split interparietal that was not associated to any other cranial malformation at 100 mg/kg/day), not dose-related and/or were within or close to the range of the Historical Control Data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Variations
See table 10.
There was no test item-related increase of the frequency of soft tissue variations.

Soft tissue variations were observed without a dose relationship (i.e absent brachiocephalic trunk), with an isolated low incidence (i.e. small kidney, dilated renal pelvis and liquid contents in cranial cavity) and/or with a litter incidence lower than controls (i.e. small gall bladder) and/or with an incidence lower than the Historical Control Data (i.e. dilated renal pelvis). Therefore they were considered to be unrelated to the test item treatment.

Other soft tissue variations, not presented in this summary table, were observed without a dose relationship and were considered to be unrelated to the test item treatment.


Malformations
See table 11.
There was no test item-related increase in the frequency of soft tissue malformations.

In the 1000 mg/kg/day group, two fetuses from two different litters showed malformations (K31094-01: marked dilated cerebral ventricle; K31080-03: ventricular septum defect and dilated aortic arch) that were not recorded in the control group and/or in the Historical Control Data.

In the 300 mg/kg/day group, three fetuses from three different litters showed malformations. Fetus K31056 03 showed marked dilated cerebral ventricle together with small cerebrum and fetus K31057-07 had small lung. Small lung is present in the Historical Control Data.
Fetus K31059-08 showed absent aortic arch and dilated pulmonary trunk. Both malformations are present in the Historical Control Data.

In the 100 mg/kg/day group, there was no visceral malformation.

In the control group, one litter contained one malformed fetus (K31007-03 with absent gall bladder).
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
other: fetal weight
Abnormalities:
effects observed, treatment-related
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Clinical signs

Dose-level (mg/kg/day)

0

100

300

1000

Emaciated appearance

0

0

0

1

Blood in the bedding

0

0

2

0

Cutaneous lesions (tail)

0

1

0

0

Abnormal growth of teeth

1

0

0

1

Difficulty during gavage administration

2

0

0

0

No remarkable observations

21

23

20

22

 

Table 2: Body weight and body weight change

Dose-level (mg/kg/day)

0

100

300

1000

Mean body weight (g)

 

 

 

 

Day 6 p.c.

3770

-

3748
(-1)

3806
(+1)

3806
(+1)

Day 29 p.c.

4128

-

4082
(-1)

4142
(0)

4015
(-3)

Mean body weight change (g)

 

 

 

 

Days 6-9 p.c.

+27

+27

+31

-2

Days 9-12 p.c.

+44

+52

+45

+47

Days 12-15 p.c.

+90

+82

+71

+54

Days 15-19 p.c.

+72

+60

+41

+64

Days 19-24 p.c.

+95

+93

+98

+40*

Days 24-29 p.c.

+30

+19

+11

+6

Days 6-29 p.c.

+358

-

+334

(-7)

+331

(-8)

+209*

(-42)

p.c.: post-coitum.

-: not applicable.

Statistical significance: *: p<0.05.

( ): in brackets, percentage difference vs. controls.


 

Table 3: Food consumption

Dose-level (mg/kg/day)

0

100

300

1000

Days 6 to 9 p.c.

177

-

169
(-5)

183
(+3)

160
(-10)

Days 9 to 12 p.c.

175

-

181
(+3)

184
(+5)

174
(-1)

Days 12 to 15 p.c.

166

-

163
(-2)

160
(-4)

142
(-14)

Days 15 to 19 p.c.

195

-

182
(-7)

174
(-11)

165
(-15)

Days 19 to 24 p.c.

173

-

168
(-3)

159
(-8)

146
(-16)

Days 24 to 29 p.c.

118

-

107
(-9)

107
(-9)

89*
(-25)

p.c.: post-coitum.

-: not applicable.

Statistical significance: *: p<0.05.

( ): in brackets, percentage difference vs. controls.

Table 4: Net body weight change

Dose-level (mg/kg/day)

0

100

300

1000

Gravid uterus weight

473.6

497.8

520.4

467.0

 

-

(+5)

(+10)

(-1)

Carcass weight

3654.3

3583.9

3621.1

3547.8

 

-

(-2)

(-1)

(-3)

Net body weight change from Day 6 p.c.

-116.1

-164.3

-189.4

-258.1

-: not applicable.

( ): in brackets, percentage difference vs. controls.


 

Table 5: Macroscopic examination

Dose-level (mg/kg/day)

0

100

300

1000

Lungs: brownish colored focus(i)

1

0

0

0

Lungs: colored nodule(s)

0

0

0

1

Abdominal cavity: liquid contents

1

0

0

1

Stomach: blackish or reddish colored depressed area(s)

0

1

0

1

Stomach: whitish colored raised area(s)

0

0

1

0

Connective tissue, periovarian region:
serous cyst(s)

3

1

1

2

Gall bladder: reduced in size

0

0

0

2

Gall bladder: bilobed

0

0

1

1

No necropsy observations

20/24

23/24

19/22

18/24

 

Table 6: Hysterectomy data

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Number of females with live fetuses
at termination

22

20

20

22

159

Mean number of corpora lutea per animal

10.4

11.9

12.3*

12.7**

[11.4-12.8]

Mean number of implantations per animal

9.0

9.9

10.9*

11.0**

[9.5-11.4]

Mean pre-implantation loss (%)

14.1

15.5

10.4

13.2

[10.5-17.9]

Mean number of live fetuses per animal

8.1

9.1

9.1

9.6

[8.5-10.6]

Dead fetuses (%)

0.0

0.5

0.0

0.0

[0.00-1.00]

Mean number of implantation scars

0.0

0.0

0.0

0.0

/

Mean number of early resorptions

0.4

0.3

0.5

0.3

/

Mean number of late resorptions

0.5

0.6

1.4**a

1.0

/

Mean post-implantation loss (%)

9.4

7.9

16.0

11.7

[3.9-20.5]

Statistical significance: *: p<0.05 and **: p<0.01.

a: statistically significant for total number of late resorptions.

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016); [Range of study means: min.; max.].

/: not reported in HCD.


 

Table 7: Fetal body weight and sex

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Mean fetal body weight

40.0

-

36.7*
(-8)

35.2**
(-12)

30.9#
(-23)

[27.6; 48.7]

Mean fetal male body weight

39.5

-

36.2

(-8)

35.5*

(-10)

31.2#

(-21)

[28.0; 50.9 ]

Mean fetal female body weight

40.0

-

37.0

(-8)

34.6**

(-14)

30.9#

(-23)

[17.2; 49.0]

Mean percentage of male fetuses

50.2

53.9

59.0

59.4

[47.4; 58.3]

-: not applicable.

Statistical significance: *: p<0.05, **: p<0.01 and #: p<0.001.

(): in brackets percentage difference vs. controls.

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016); [Range of litter means: min.; max.].

Table 8: External variations

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Litters affected, n (%)

2 (9.1)

2 (10.0)

3 (15.0)

7 (31.8)

11 (6.9)(b)

Fetuses affected, n (%)

2 (1.1)

2 (1.1)

3 (1.6)

15** (7.1)

14 (0.9)(b)

Amniotic fluid: abnormal color, F (L) %

0 (0)

0 (0)

0 (0)

5.7#(22.7*)

0 (0)

Local edema,F (L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0 (0)

Paw: hyperflexion,F (L) %

1.1 (9.1)

1.1 (10.0)

1.1 (10.0)

2.4 (22.7)

0 (0)

F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

Statistical significance: *: p<0.05, **: p<0.01 and #: p<0.001 for number of fetuses or litters.

(b): mean incidence.


 

Table 9: External malformations

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Litters affected, n (%)

0 (0)

1 (5.0)

0 (0)

3 (13.6)

6 (3.8)(b)

Fetus affected, n (%)

0 (0)

1 (0.6)

0 (0)

3 (1.4)

6 (0.4)(b)

Affected Fetuses/Litter, Mean%

0.0

0.6

0.0

5.4

/

Cleft lip, F (L) %

0 (0)

0.6 (5.0)

0 (0)

0 (0)

0 (0)

Digit: ectrodactyly,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Digit: malpositioned,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Trunk: gastroschisis,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0.5 (5.3)(a)

Trunk: omphalocele,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Trunk: ombilical hernia,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0.4 (4.0)(a)

F: fetal incidence, L: litter incidence.

/:not reported in HCD; HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence; (b): mean incidence.

Table 10: Soft tissue variations

Dose-level (mg/kg/day)

0

100

300

1000

HCD(a)

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Cranial cavity: liquid contents, F (L) %

0 (0)

0 (0)

0.5 (5.0)

0.5 (4.5)

0 (0)

Gall bladder: small, F (L) %

2.2 (18.2)

1.1 (10.0)

1.1 (5.0)

4.2 (13.6)

0.6 (5.6)

Kidney: dilated renal pelvis,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

1.7 (16.0)

Kidney: small kidney,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Brachiocephalic trunk: absent,F (L) %

0 (0)

2.2 (15.0)

1.1(10.0)

2.4 (13.6)

4.0 (29.2)

F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence.


 

Table 11: Soft tissue malformations

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Litters affected, n (%)

1 (4.5)

0 (0)

3 (15.0)

2 (9.1)

8 (5.0)(b)

Fetus affected, n (%)

1 (0.6)

0 (0)

3 (1.6)

2 (0.9)

8 (0.5)(b)

Affected Fetuses/Litter, Mean%

0.8

0.0

2.1

5.3

/

Brain: small cerebrum, F (L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0 (0)

Brain: marked dilated cerebral ventricle,F (L) %

0 (0)

0 (0)

0.5 (5.0)

0.5 (4.5)

0 (0)

Heart: ventricular septum defect,

 F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Lungs: small,F (L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0 (0)

Gall bladder: absent,F (L) %

0.6 (4.5)

0 (0)

0 (0)

0 (0)

0.6 (5.6)(a)

Vessels: dilated aortic arch,F (L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0.4 (4.0)(a)

Vessels: absent aortic arch, F (L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0.5 (5.3)(a)

Vessels: dilated pulmonary trunk,

F (L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0.5 (5.3)(a)

F: fetal incidence, L: litter incidence.

/:not reported in HCD; HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence; (b): mean incidence.


 

Table 12: Skeletal examinations

Cartilage

Dose-level (mg/kg/day)

0

100

300

1000

HCD(a)

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Cartilage of sternebra(e): present, F(L) %

34.1 (81.8)

41.7 (85.0)

47.8*(95.0)

49.1**(95.5)

15.7 (62.5)

Cartilage of sternebra(e): misshapen, F(L) %

0 (0)

0 (0)

0 (0)

1.4 (9.1)

0 (0)

Cartilage of rib(s): branched, F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Cartilage of metacarpal bone(s): present,F(L) %

6.7 (27.3)

7.2 (50.0)

9.9 (55.0)

20.3#(54.5)

4.5 (33.3)

Forepaw: cartilage of median phalanx present,F(L) %

4.5 (22.7)

15.0**(55.0)

6.6 (50.0)

12.3**(45.5)

6.3 (29.2)

Cartilage of talus: present,

F(L) %

0 (0)

0 (0)

1.1 (10.0)

4.7** (31.8**)

0 (0)

Cartilage of metatarsal bone absent,F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence.

Statistical significance: *: p<0.05 , **: p<0.01, #: p<0.001 for number of fetuses or litters.

Table 13: Skeletal variations

Dose-level (mg/kg/day)

0

100

300

1000

HCD(a)

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Interparietal: incomplete ossification, F(L) %

1.1 (9.1)

4.4 (30.0)

4.4 (35.0)

4.7*(36.4)

4.1 (36.8)

Hemisternebra(e): unossified,

F(L) %

2.8 (13.6)

1.7 (15.0)

5.5 (30.0)

6.1 (50.0*)

2.9 (16.7))

Hemisternebra(e): incomplete ossification, F(L) %

2.2 (9.1)

1.7 (15.0)

4.9 (30.0)

6.6 (50.0**)

3.5 (22.2)

6thsternebra(e): incomplete ossification, F(L) %

2.2 (18.2)

2.8 (20.0)

4.4 (30.0)

8.5**(27.3)

6.9 (42.1)

6thsternebra(e): unossified, F(L) %

0 (0)

0 (0)

0.5 (5.0)

0.9 (9.1)

0.4 (4.2)

Supernumerary 13thrib(s), F(L) %

50.8 (86.4)

70.6#(100.0)

65.4**(100.0)

62.7*(90.9)

81.2 (100.0)

Knobby rib(s), F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Supernumerary 14thrib(s), F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Metacarpal bone: incomplete ossification,1stmetacarpal(s),F(L) %

5.6 (22.7)

6.1 (40.0)

6.6 (35.0)

14.2**(45.5)

8.4 (61.1)

Metacarpal bone: unossified
1stmetacarpal(s),F(L) %

1.1 (9.1)

2.2 (20.0)

3.3 (30.0)

9.4#(45.5*)

7.4 (33.3)

Forepaw: incomplete ossification median phalanx,F(L) %

3.4 (22.7)

10.6*(45.0)

6.0 (40.0)

9.4*(40.9)

10.0 (44.0)

Forepaw: unossified median phalanx,F(L) %

1.1 (9.1)

5.6*(30.0)

2.2 (15.0)

4.2 (22.7)

3.8 (20.8)

Talus: unossified, F(L) %

0 (0)

0 (0)

0.5 (5.0)

2.4 (18.2)

0.5 (5.6)

Talus: incomplete ossification, F(L) %

0 (0)

0 (0)

0.5 (5.0)

2.8*(22.7*)

0.6 (5.6)

HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence.

Statistical significance: *: p<0.05, **: p<0.01, #: p<0.001 for number of fetuses or litters.

Table 14 : Skeletal malformations

Dose-level (mg/kg/day)

100

300

1000

HCD

Dams with live fetuses, n

22

20

20

22

159

Live fetuses, n

179

180

182

212

1558

Litters affected, n (%)

8 (36.4)

4 (20.0)

10 (50.0)

15 (68.2)

46 (28.9)(b)

Fetus affected, n (%)

10 (5.6)

4 (2.2)

15 (8.2)

21 (9.9)

56 (3.6)b)

Affected Fetuses/Litter, Mean%

6.4

2.5

7.7

13.7

/

Supraoccipital: split,F(L) %

0.6 (4.5)

0 (0)

0 (0)

0 (0)

0 (0)

Interparietal: split,F(L) %

1.1 (9.1)

1.7 (15.0)

2.2 (15.0)

2.8 (27.3)

1.2 (11.1)(a)

Interparietal: absent,F(L) %

1.7 (13.6)

0 (0)

1.6 (15.0)

0.9 (4.5)

3.4 (31.6)(a)

Lumbar hemivertebra:
supernumerary,F(L) %

0.6 (4.5)

0 (0)

0 (0)

0 (0)

0 (0)

Spine: scoliosisF(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Sternebra(e): fused,F(L) %

2.2 (13.6)

0.6 (5.0)

3.8 (30.0)

5.7 (45.5*)

2.2 (20.8)(a)

Rib(s): fused,F(L) %

0 (0)

0 (0)

0.5 (5.0)

0 (0)

0.9 (8.3)(a)

Tibia: misshapen,F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Metatarsal(s): absent, F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

Hindpaw: absent digit(s),

 F(L) %

0 (0)

0 (0)

0 (0)

0.5 (4.5)

0 (0)

/:not reported in HCD; HCD: Historical Control Data (NZW rabbits, June 2015 to September 2016).

(a): maximal incidence; (b): mean incidence.

Statistical significance: *: p<0.05 for number of litters.

Table 15 : Pregnancy status

Dose-level (mg/kg/day)

0

100

300

1000

Mated dams, n

24

24

24

24

Pregnant females alive at term, n

22

20

20

22

Non pregnant dams, n

2

4

2

2

Prematurely euthanized dams (poor clinical condition), n

0

0

1

0

Found dead dams, n

0

0

0

0

Aborted dams, n

0

0

1

0

Dams with live fetuses on Day 29p.c.

22

20

20

22

n: number

Conclusions:
The test item, Propylidynetrimethyl trimethacrylate, was administered by gavage, once daily from Days 6 to 28 p.c. inclusive, to mated female New Zealand White rabbits at dose levels of 100, 300 or 1000 mg/kg/day.

According to the results obtained in this study:
-The No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day based on the lower body weight gain, lower net body weight change and lower food consumption observed at 1000 mg/kg/day.
-The NOAEL for embryo-fetal development was considered to be 300 mg/kg/day based on adverse effects on fetal weight associated with increased incidences of external malformations at 1000 mg/kg/day.
Executive summary:

The objective of this prenatal developmental toxicity 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 rabbits from implantation to the day before scheduled hysterectomy [Day 6 to Day 28 post-coitum (p.c.) inclusive].

Methods

The test item was administered to three groups of 24 mated female New Zealand White rabbits, once daily from Day 6 to Day 28 p.c., by gavage, at dose levels of 100, 300 or 1000 mg/kg/day (groups 2 to 4). An additional group of 24 mated females received the vehicle, 0.5% Carboxymethylcellulose (400-800 cps) / 0.5% Tween 80 in drinking water treated by reverse osmosis, under the same experimental conditions, and acted as the control group (group 1). A constant dosage volume of 3 mL/kg/day was used.

 

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c. animals were euthanized and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. Macroscopic lesions were sampled and preserved in a fixative. Live fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.

 

Results

Chemical analysis of dose formulations: the test item concentrations in the administered dose formulations analyzed infirst and last weeks of treatmentremained within an acceptable range of variations (-1.3% to +8.0%) when compared with the nominal values (± 15% of the nominal concentrations).

 

Pregnancy status: in the control, 100, 300 and 1000 mg/kg/day groups, there were 22, 20, 20 and 22 females with live fetuses at hysterectomy, respectively.

 

Mortality/abortion: there were no test item-related unscheduled deaths or abortions in any group.

 

Clinical signs: there were no remarkable clinical observations in surviving animals.

 

Body weight, body weight change, net body weight change and food consumption:at 1000 mg/kg/day and when compared with controls, there were lower body weight change over the whole treatment period (-42%, p<0.05),net body weight change on Day 29p.c.(-258.1 gvs. -116.1 g in control animals)and food consumption (down to -25% on Days 24 to 29p.c., p<0.05).During the gestation period (from Day 6p.c.to Day 29p.c.), four out of twenty-two females showed body weight loss together with decreased food consumption and low net body weight change values.

Taking together, these findings were considered to be adverse at 1000 mg/kg/day.


Maternal terminal examination: at necropsy, discolored area(s) were observed in the stomach of one female in each dose levels group.Taking into account the absence of similar findings in the control group, a test item treatment relationship cannot be excluded.

There were no effects on mean gravid uterus weight or mean carcass weight.

 

Hysterectomy data: there were no test item-related effects on hysterectomy data in surviving dams on Day 29p.c.

 

Fetal body weight and percentage of male fetuses:

From 100 mg/kg and when compared with controls, lower mean fetal boy weights were observed but all values were within the HCD. At 1000 mg/kg/day, the mean litter value of fetal body weight was decreased when compared with controls and outside the HCD. This decrease at 1000 mg/kg/day was considered as adverse.

 

At 300 and 1000 mg/kg/day, a minimally, not statistically significant, higher percentage of male fetuses was observed (59.0% and 59.4%, respectively,vs.50.2% in controls). This non-adverse percentage was close to the upper limit of the Historical Control Data (58.3%).

 

Fetal examinations:

External examination

At 1000 mg/kg/day and when compared with controls, there were test item-related higher fetal and litter incidences of external variations, namely abnormal color of amniotic fluid [5.7% (p<0.001) and 22.7% (p<0.05)vs.0% in controls, respectively].

Three fetuses from three different litters also had ectrodactyly, malpositioned digit and gastroschisis (fetus K31086-01 F), omphalocele (fetus K31083-11 M) and umbilical hernia (fetus K31094-01 M). Although the fetal and litter incidences of these malformations mainly impacting the abdominal wall were similar to the Historical Control Data, a relationship to the test item cannot be ruled out.

 

Soft tissue examination

There were no test item-related variations or malformations at soft tissue examinationat any dose level.

 

Cartilage and skeletal examinations

When compared with controls and/or Historical Control Data, test item-related findings of delayed ossification were noted with higher litter and/or fetal incidences from 100 mg/kg/day [i.e.unossification of 1st metacarpal bone and/or incomplete ossification of hemisternebra(e)], from300 mg/kg/day [i.e.incomplete ossification of interparietal,unossification of hemisternebra(e), and/or incomplete ossification of 6th sternebra(e)] and at 1000 mg/kg/day[i.e.unossification of 6th sternebra(e), incomplete ossification of 1st metacarpal bone and/or unossification or incomplete ossification of talus].

 

When compared with controls and/or Historical Control Data, there were increased fetal and litter incidences of fetuses with fused sternebra(e) at 300 (3.8% and 30.0%vs.2.2% and 13.6% in controls, respectively) and 1000 mg/kg/day [5.7 and 45.5 (p<0.05), respectively], and split interparietalat 300 (2.2% and 15.0%vs.1.1% and 9.1% in controls, respectively) and1000 mg/kg/day (2.8% and 27.3%).These test item-related findings were rather considered as variations (non-adverse findings) asthey were consistent with a developmental delay in ossification.


Onefetus (K31086-01 F), showing external malformations, also had skeletal malformations (tibia misshapen, absent digits hindpaw and scoliosis).

 

Conclusion

The test item,Propylidynetrimethyl trimethacrylate, was administered by gavage, once daily from Days 6 to 28 p.c.inclusive, to mated female New Zealand White rabbits at dose levels of 100, 300 or 1000 mg/kg/day.

 

According to the results obtained in this study:

.         The No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day based on the lower body weight gain, lower net body weight change and lower food consumption observed at 1000 mg/kg/day.

.         The NOAEL for embryo-fetal development was considered to be 300 mg/kg/day based on adverse effects on fetal weight associated with increased incidences of external malformations at 1000 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
This study is reliable, performed with OECD guidelines.
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

First key study: Developmental study in rats (Braun 2015):

The oral (gavage) prenatal developmental toxicity of the test item Propylidynetrimethyl trimethacrylate (CAS 3290-92-4) to Wistar rats was investigated in a GLP-compliant dose-effect study according to the OECD TG 414 (2001) guideline.

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to caesarean section). Four groups of 24 mated females per group were treated by gavage once daily at nominal dose levels of 0 (control group), 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively). A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil). All females were sacrificed on day 21 post coitum and the foetuses were removed by caesarean section.

All females survived until the scheduled necropsy. No clinical signs were recorded in any group, and the mean daily food consumption of all groups compared favourably. Although the mean absolute body weights were unaffected, a statistically significant lower mean body weight gain of the dams in group 4 (40 vs. 46 % in the controls on day 21 post coitum) and a statistically significant decreased corrected body weight gain (corrected for the gravid uterus weight) in group 4 (8.0 vs. 11.5 % in the controls on day 21) were considered to be test item-related. The reproduction data (post-implantation loss and mean number of foetuses per dam) was unaffected by treatment and no macroscopical findings were noted at any dose level.

The mean placenta weights of all groups were similar. The external examination of the foetuses showed no abnormalities of toxicological relevance and sex ratios were unaffected. Foetuses of group 4 dams had lower mean body weights (-6 % on litter basis and -8 % on individual basis). This was considered to be related to the treatment with test item.

In conclusion based on the slightly lower mean body weight gain, the NOEL (No Observed Effect Level) for maternal toxicity was considered to be 300 mg/kg bw/day, whereas the NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be 1000 mg/kg bw/day or higher. For prenatal development, the NOEL (No Observed Effect Level) and the NOAEL (No Observed Adverse Effect Level) were considered to be 300 mg/kg/day, based on the lower mean foetal weights noted at 1000 mg/kg/day.

First supporting study: Dose-range finding study in rats (Kaiser 2014):

The oral (gavage) prenatal developmental toxicity of the test item Propylidynetrimethyl trimethacrylate (CAS 3290-92-4) to Wistar rats was investigated in a non-GLP dose range-finding study similar to the OECD TG 414 (2001) protocol.

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to caesarean section). Four groups of 8 mated females per group were treated by gavage with the test item once daily at nominal dose levels of 0 (control group), 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively). Using corn oil as vehicle a standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was administered. Control animals were dosed with the same volume of vehicle alone.

The treatment with the test item did not result in any mortalities. No clinical signs were observed. No effects on food consumption were recorded. At necropsy, no macroscopic findings related to the treatment with the test item were found.

No findings were noted during external examination of foetuses. No effects on sex ratio and body weights of foetuses were recorded.

In group 4, the body weight gain and corrected body weight gain of the dams was slightly decreased. The post-implantation loss and the number of embryonic resorptions were slightly increased. The number of foetuses (in relation to the number of implantations) was slightly decreased. These effects were considered test item-related. No effects of the treatment with the test item were recorded in groups 2 and 3.

In conclusion based on the results of this study, nominal dose levels of 0, 100, 300 and 1000 mg/kg bw/day are considered to be suitable for the subsequent main study for effects on embryo-foetal development in the Han Wistar rat.

Second supporting study : developmental study in rats (Serota 1982):

Test compound (TMPTMA) was evaluated for tolerance in pregnant rats to determine dose levels for a subsequent teratology screening study. Test substance was administered by gavage at 500, 2500 and 5000 mg/kg/day (6 inseminated females per dose) from Days 6-15 of gestation. The dose of 5000 mg/kg/day was highly toxic for females; mortality, severe clinical signs, decrease of bodyweight and macroscopic changes were observed. As the dose of 2500 mg/kg/day was well tolerated by females, this dose was selected to evaluate the embryo/fetal toxicity and teratogenic effects of Propylidynetrimethyl trimethacrylate. Test substance was administered by gavage from Days 6-15 of gestation to 22 females.

Compound-related maternal toxicity was observed: decreased survival, decreased body weight gains, increased incidence of clinical signs, and an increased incidence of gross pathology findings. Foetotoxic effects were observed and consisted of effects on resorptions, fetal viability, fetal weights and fetal lengths. The decrease in mean gravid uterine weights was directly attributed to the foetotoxic effect. Those effects on the fetuses considered to be directly related to the maternal toxicity of the test compound. An increase in the incidence of visceral variants (23%) was observed when compared to control (3%). A higher than control number of fetuses exhibiting skeletal variants was observed. The incidence was found to be significant for skeletal variants, lagging ossification, skull closure less than 75% and angulated ribs. The types of visceral and skeletal variants observed to be increased over control are suggestive as resulting from the maternal toxicity noted.

The Registrants consider that it is not possible to conclude on the developmental toxicity of Propylidynetrimethyl trimethacrylate in rats based on Serota’s studies based on the following arguments: Only one group tested at the maximum tolerated dose of 2500 mg/kg/day; High incidence of maternal death (5/22 females); Severe decreased maternal body weight during days 6-20 of pregnancy (-20 g for treated females vs +87 g in controls); Only 17 females evaluated for fetal examinations in a context of severe maternal toxicity; No NOAEL could be set.

Second key study: Developmental study in rabbits (Papineau 2018):

The objective of this prenatal developmental toxicity 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 rabbits from implantation to the day before scheduled hysterectomy [Day 6 to Day 28 post-coitum (p.c.) inclusive]. The test item was administered to three groups of 24 mated female New Zealand White rabbits, once daily from Day 6 to Day 28p.c., by gavage, at dose levels of 100, 300 or 1000 mg/kg/day (groups 2 to 4). An additional group of 24 mated females received the vehicle, 0.5% Carboxymethylcellulose (400-800 cps) / 0.5% Tween 80 in drinking water treated by reverse osmosis, under the same experimental conditions, and acted as the control group (group 1).A constant dosage volume of 3 mL/kg/day was used.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29p.c.animals were euthanized and submitted to a macroscopicpost-mortemexamination. Hysterectomy was performed and the numbers ofcorpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. Macroscopic lesions were sampled and preserved in a fixative. Live fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.

In the control, 100, 300 and 1000 mg/kg/day groups, there were 22, 20, 20 and 22 females with live fetuses at hysterectomy, respectively.

There were no test item-related unscheduled deaths or abortions in any group. There were no remarkable clinical observations in surviving animals.

At 1000 mg/kg/day and when compared with controls, there were lower body weight change over the whole treatment period (-42%, p<0.05),net body weight change on Day 29p.c.(-258.1 gvs. -116.1 g in control animals)and food consumption (down to -25% on Days 24 to 29p.c., p<0.05).During the gestation period (from Day 6p.c.to Day 29p.c.), four out of twenty-two females showed body weight loss together with decreased food consumption and low net body weight change values. Taking together, these findings were considered to be adverse at 1000 mg/kg/day.

At necropsy, discolored area(s) were observed in the stomach of one female in each dose levels group. Taking into account the absence of similar findings in the control group, a test item treatment relationship cannot be excluded.

There were no effects on mean gravid uterus weight or mean carcass weight. There were no test item-related effects on hysterectomy data in surviving dams on Day 29p.c.

From 100 mg/kg and when compared with controls, lower mean fetal boy weights were observed but all values were within the HCD. At 1000 mg/kg/day, the mean litter value of fetal body weight was decreased when compared with controls and outside the HCD. This decrease at 1000 mg/kg/day was considered as adverse.

At 300 and 1000 mg/kg/day, a minimally, not statistically significant, higher percentage of male fetuses was observed (59.0% and 59.4%, respectively, vs. 50.2% in controls). This non-adverse percentage was close to the upper limit of the Historical Control Data (58.3%).

At 1000 mg/kg/day and when compared with controls, there were test item-related higher fetal and litter incidences of external variations, namely abnormal color of amniotic fluid [5.7% (p<0.001) and 22.7% (p<0.05)vs.0% in controls, respectively].

Three fetuses from three different litters also had ectrodactyly, malpositioned digit and gastroschisis (fetus K31086-01 F), omphalocele (fetus K31083-11 M) and umbilical hernia (fetus K31094-01 M). Although the fetal and litter incidences of these malformations mainly impacting the abdominal wall were similar to the Historical Control Data, a relationship to the test item cannot be ruled out.

There were no test item-related variations or malformations at soft tissue examinationat any dose level.

When compared with controls and/or Historical Control Data, test item-related findings of delayed ossification were noted with higher litter and/or fetal incidences from 100 mg/kg/day [i.e.unossification of 1st metacarpal bone and/or incomplete ossification of hemisternebra(e)], from300 mg/kg/day [i.e.incomplete ossification of interparietal,unossification of hemisternebra(e), and/or incomplete ossification of 6th sternebra(e)] and at 1000 mg/kg/day [i.e.unossification of 6th sternebra(e), incomplete ossification of 1st metacarpal bone and/or unossification or incomplete ossification of talus].

When compared with controls and/or Historical Control Data, there were increased fetal and litter incidences of fetuses with fused sternebra(e) at 300 (3.8% and 30.0%vs.2.2% and 13.6% in controls, respectively) and 1000 mg/kg/day [5.7 and 45.5 (p<0.05), respectively], and split interparietalat 300 (2.2% and 15.0%vs.1.1% and 9.1% in controls, respectively) and1000 mg/kg/day (2.8% and 27.3%).These test item-related findings were rather considered as variations (non-adverse findings) asthey were consistent with a developmental delay in ossification. One fetus (K31086-01 F), showing external malformations, also had skeletal malformations (tibia misshapen, absent digits hindpaw and scoliosis).

In conclusion, According to the results obtained in this study:  The No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day based on the lower body weight gain, lower net body weight change and lower food consumption observed at 1000 mg/kg/day. The NOAEL for embryo-fetal development was considered to be 300 mg/kg/day based on adverse effects on fetal weight associated with increased incidences of external malformations at 1000 mg/kg/day.

Third supporting study : dose-rande finding study in rabbits (Chalmey 2017):

The test item, was administered to time-mated female New-Zealand White rabbits, by oral route, once daily, from Days 6 to 28p.c., at dose levels of 100, 300 or 1000 mg/kg/day.

At 100 mg/kg/day, there were no adverse findings on maternal parameters orat examination of the fetuses.

At 300 mg/kg/day, there were no toxicity findings on maternal parameters; the mean fetal body weight was reduced when compared with controls.

At 1000 mg/kg/day, in average, the females lost weight between Days 6 and 9p.c.and the mean food consumption was slightly reduced in dams. However, there were no effects on dam mean body weight. The fetal body weight was reduced when compared with controls.

Therefore, on the basis of the experimental conditions and results of this study, the dose of 1000 mg/kg/day was considered to be below the Maximum Tolerated Dose in pregnant rabbits and could be selected as the highest dose to be tested in a further main study.

Justification for classification or non-classification

Based on the available data, no classification for reproduction is required for TMPTMA according to the CLP Regulation (EC) N° 1272-2008. An additional study (OECD 443) is proposed in this dossier in order to confirm the absence of classification for reproduction parameters.