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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-10-19 to 2004-12-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guidceline study, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test), adopted on 22 March 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA guideline OPPTS 870.3650, July 2000
Deviations:
no
Principles of method if other than guideline:
The study was conducted as a combined repeated dose toxicity study with Reproduction / Developmental Toxicity Screening Test. In this part all parameters concerning Reproduction / Developmental Toxicity were considered.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: males were 8 weeks old and the females were 10 weeks old
- Weight at study initiation: 303 g (range: 280 g to 332 g) for the males and 229 g (range: 199 g to 273 g) for the females
- Fasting period before study: no
- Housing: The animals were housed individually (except during mating) in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust was placed under each cage. Shortly before parturition, the females were moved to polycarbonate cages (43.0 x 21.5 x 20.0 cm) with autoclaved sawdust and wood shavings as nesting material (SICSA, Alfortville, France).
- Diet: The animals had free access to SSNIFF R/M-H pelleted maintenance diet, Batch Nos. 2764132 and 4764439 (SNIFF Spezialdiäten GmbH, Soest, Germany) distributed weekly.
- Water: The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: 7 days

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

IN-LIFE DATES: From: 2004-10-12 To: 2004-12-07

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Vehicle
The vehicle was corn oil, Batch No. 103K0107, supplied by Sigma (Saint-Quentin-Fallavier, France).

Dosage formulation preparation
The test item dosage formulations were prepared by suspending Allyl Methacrylate (AMA) in corn oil in order to achieve the concentrations of 0.6, 3 and 12 mg/mL and were stored at +4°C or up to 9 days protected from light.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis of the dosage formulations
Homogeneity
Two dosage formulations were prepared at 0.2 and 15 mg/mL of AMA (in corn oil) in order to cover all the concentrations intended for use in this study. From both dosage formulations, duplicate samples were taken at three different levels of the container (top, middle, bottom) and analyzed for concentration of the test item just after preparation (day 0) and after 9 days of storage at +4°C (protected from light).
Stability
The two dosage formulations prepared at 0.2 and 15 mg/mL of AMA in corn oil for homogeneity analysis were sampled after 0 (just after preparation), 4 and 9 days storage at +4°C (protected from light). The aliquots (n = 2) sampled on day 4 were stored frozen at -20°C pending analysis on the last sampling occasion, when all samples were assayed. The mean concentrations measured on days 0 and 9 for homogeneity were taken as the initial and final values for the stability test.
Concentration
The concentration of samples taken from each dosage formulation (including the control) prepared for use in weeks 1, 2, 4 and 7 was determined.

Results
A satisfactory agreement was observed between the nominal and actual concentrations of the test item in the dosage formulations analyzed since the deviations from nominal concentration remained in an acceptable range of ± 10%. Furthermore, there was a satisfactory correspondence between the nominal and the measured concentrations of the test item in the vehicle.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: during the 2 week mating period, one male and one female from the same dose group were co-habitated during the night until pregnancy occurs or the mating period is over
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
Duration of treatment / exposure:
Females: 15 days before mating, through mating period, pregnancy and lactation, until day 5 post-partum inclusive (or until sacrifice, for unmated females);
Males: 15 days before mating, through mating and post-mating periods through sacrifice (minimum 4 weeks);
Frequency of treatment:
once daily
Duration of test:
Males: 15 days before mating, through mating and post-mating periods through sacrifice (minimum 4 weeks);
Females: 15 days before mating, through mating period, pregnancy and lactation, until day 5 post-partum inclusive (or until sacrifice, for unmated females);
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected following the results of a previously conducted study CIT/Study No. 28198 TSR in which rats were dosed (gavage) withAllyl Methacrylate (AMA), Batch. 00216X, at 0, 25, 75 and 150 mg/kg/day for 10 consecutive days. In this study, 150 mg/kg/day resulted in morbidity and mortality, both sexes in the 25 and 75 mg/kg/day groups showed signs of hypersalivation during the dosing period, males showed signs of piloerection at 75 mg/kg/day, and the females in the 75 mg/kg/day group had increased liver weight.
Taking into account that the duration of the dosing period for this study was nearly 6 weeks in females, the high dose-level was set at 60 mg/kg/day. The intermediate and low dose-levels were selected in order to identify a No Effect Level.

Examinations

Maternal examinations:
- CLINICAL OBSERVATIONS: Clinical observations were conducted daily throughout the study period.

- DETAILE CLINICAL OBSERVATIONS (DCO): Conducted on all rats pre-exposure and weekly throughout the study. The DCOs included cage-side, handheld and open-field observations that were recorded categorically or using explicitly defined scales.

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded weekly throughout the study. Female body weights were performed weekly for the premating and mating periods. Maternal body weights were recorded on gd 0, 7, 14, 17 and 20. Females that delivered were weighed on ld 1 and 5. Females that failed to mate or deliver were weighed on a weekly basis for the study duration.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined weekly for males and females during the pre-mating period. Due to co-housing, food consumption was not measured during mating. During gestation, food consumption for the females was measured on gd 0, 7, 10, 14, 17 and 20. After parturition, food consumption was measured on ld 1 and 5.

OTHER:
Females were observed for signs of parturition beginning on gd 20. The first day the presence of a litter was noted was designated ld 1.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
- Number of implantations: Yes
Fetal examinations:
All litters were examined as soon as possible after delivery and the following information was recorded: litter size, the number of live and dead pups and the sex. Pup clinical observations and body weights were recorded on pnd 1 and 5. In addition, any physical abnormalities or demeanor changes were recorded during the lactation period.
Pups found dead and sacrificed on ld 6 were carefully examined for external gross abnormalities and a macroscopic evaluation was performed. All litters were examined as soon as possible after delivery.
Statistics:
The following sequence was used for the statistical analyses of body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data. If there were 3 or less animals/sex/group remaining, no statistical analysis was performed. If there were 5 or less animals/sex/group remaining for 3 or more groups, the data were analyzed using the Dunn test. If there was 5 or less animals/sex/group remaining for less than 3 groups, the data were analyzed using the Mann-Whitney/Wilcoxon test. If there was 5 or more animals/sex/group, the data were analyzed by Kolmogorov-Lillefors test to test for the normality of the distribution of the data. If it was not normal, the values were logarithmically transformed and retested for normality. If it still did not meet the criteria for normality, the original values were analyzed by the Dunn test. If the data were normal, and there were 3 or more groups, the data were assessed for homogeneity of variance between groups with the Bartlett test. If not homogeneous, the data were analyzed by the Dunn test; if homogeneous, the data were assessed by the Dunnett test. If the data were normal, and there was less than 3 groups, the data were assessed for homogeneity of variance between groups with the Fisher test. If not homogeneous, the data were assessed using the Mann-Whitney/Wilcoxon test; if homogeneous, the data were analyzed by the Student test. The statistical comparison of results of motor activity (horizontal andrearing movements recorded from five males and five females per group) was performed, according to the following steps: step 1: normality and homogeneity of variances (Kolmogorov-Smirnov and Bartlett test respectively), step 2: parametric (ANOVA plus Dunnett test if necessary) or non-parametric (Kruskal-Wallis plus Dunn test if necessary) tests was conducted based on the results obtained at step 1. These statistics were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc).
Indices:
The following calculations were made:
- Pre-implantation loss
- Post-implantation loss
- Mating index
- Fertility index
- Gestation index
- Live birth index
- Viability index
Historical control data:
no data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hypersalivation was observed in a dose-related manner in males and females given 15 or 60 mg/kg/day during the premating period (from week 2) and then in females during gestation and lactation. This clinical sign was not considered to be adverse as it likely represents a reaction to the dosing procedure and not a direct effect of AMA toxicity. At 60 mg/kg/day, one female experienced hypotonia and half-closed eyes on day 5 p.p.. There was no disturbance of autonomal or physiological functions at any dose-levels and the motor activity of the animals was considered to be unaffected by treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on group mean male body weight gain during the premating phase and only a marginal reduction in body weight gain was observed at 15 and 60 mg/kg/day (p<0.05) during the mating period
Haematological findings:
no effects observed
Description (incidence and severity):
There were no changes in hematological parameters that could be considered toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For blood biochemistry investigation, one female dosed with 60 mg/kg/day had increased total bilirubin concentration and another one had increased biliary acid concentration, these changes were correlated with histopathological findings in the liver
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no effects of treatment on pH, specific gravity or volume of urine produced by the males in any group.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The necropsy of the adult males and females revealed the presence of yellowish areas in the liver of 2/5 females in the 60 mg/kg/day dose group.
At histopathology, several foci of degenerated/necrotic hepatocytes, together with slight periportal fibrosis, slight biliary proliferation, and greenish-pigment-laden macrophages, were noted in 3/5 females given 60 mg/kg/day. There were no other treatment-related histopathological changes for either sex at any dose.

Maternal developmental toxicity

Dead fetuses:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the mean number of liveborn pups or on pup death after birth.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The duration of gestation was similar between the control and test item-treated groups and close to the normal value of 21 days
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There was no effect of treatment on mating at any dose-level.
The male and female fertility indices were unaffected by treatment; all mated females were pregnant with live fetuses
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on body weights of females during the premating period, gestation or lactation.
There were no external abnormalities in the control or test item-treated groups.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Hypersalivation was observed in a dose-related manner in males and females given 15 or 60 mg/kg/day during the premating period (from week 2) and then in females during gestation and lactation. This clinical sign was not considered to be adverse as it likely represents a reaction to the dosing procedure and not a direct effect of AMA toxicity. At 60 mg/kg/day, one female experienced hypotonia and half-closed eyes on day 5 p.p.. There was no disturbance of autonomal or physiological functions at any dose-levels and the motor activity of the animals was considered to be unaffected by treatment. There was no effect of treatment on group mean male body weight gain during the premating phase and only a marginal reduction in body weight gain was observed at 15 and 60 mg/kg/day (p<0.05) during the mating period. There was no effect of treatment on body weights of females during the premating period, gestation or lactation. There was no effect of treatment on food consumption for females during the premating period, gestation or lactation. There were no changes in hematological parameters that could be considered toxicologically relevant.
For blood biochemistry investigation, one female dosed with 60 mg/kg/day had increased total bilirubin concentration and another one had increased biliary acid concentration, these changes were correlated with histopathological findings in the liver. There were no effects of treatment on pH, specific gravity or volume of urine produced by the males in any group.
-Mating Data: There was no effect of treatment on mating at any dose-level.
-Fertility Data: The male and female fertility indices were unaffected by treatment; all mated females were pregnant with live fetuses.
-Delivery Data: The duration of gestation was similar between the control and test item-treated groups and close to the normal value of 21 days. There was no effect of treatment on the mean number of liveborn pups or on pup death after birth. There were no external abnormalities in the control or test item-treated groups.
The necropsy of the adult males and females revealed the presence of yellowish areas in the liver of 2/5 females in the 60 mg/kg/day dose group.
At histopathology, several foci of degenerated/necrotic hepatocytes, together with slight periportal fibrosis, slight biliary proliferation, and greenish-pigment-laden macrophages, were noted in 3/5 females given 60 mg/kg/day. There were no other treatment-related histopathological changes for either sex at any dose.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no effect of treatment on the mean number of liveborn pups or on pup death after birth.
There were no external abnormalities in the control or test item-treated groups.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: No relevant findings were observed in the pups sacrificed on day 6 postpartum

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In this study, the NOAEL for developmental toxicity was 60 mg/kg bw/day (the highest dose tested).
Executive summary:

In this combined repeated-dose toxicity study on Allyl methacrylate with reproduction/developmental screening according to OECD TG 422, there were no effects on mean number of live born pups or pup death after birth. Male and female pups at 15 or 60 mg/kg bw/day gained less weight than the controls but the changes did not reach statistical significance. The number of pups that were cold to the touch was higher at 60 mg/kg bw/day than in the control litters. No relevant findings were observed in the pups sacrificed on day 6 postpartum. Based on liver effects the NOAEL for maternal toxicity was 15 mg/kg bw/day. The NOAEL for developmental toxicity was 60 mg/kg bw/day (the highest dose tested).