Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
06-01-1990 until 11-11-1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with national standard methods with acceptable restrictions, GLP.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1992
Title:
The reproductive and neural toxicities of acrylamide and three analogues in swiss mice, evaluated using the continuous breeding protocol.
Author:
Chapin RE, Fail PA, George JD, Grizzle TB, Heindle JJ, Harry GJ, Collins BJ, Teague J
Year:
1995
Bibliographic source:
Fundamental and Apllied Toxicology Vol. 27: 9-24
Reference Type:
publication
Title:
Unnamed
Year:
1997

Materials and methods

Principles of method if other than guideline:
Method: modified reproductive assessment continuous breeding protocol (RACB)
Task 1: Range-finding study (28 days treatment), doses of Methacrylamide ranging from 0 to 720 ppm in drinking water
At day 21 of treatment, the animals were housed in breeding pairs within dose group. Ability of the pairs to mate was evaluated by checking the females for vaginal copulatory plugs each day during cohabitation (seven days). At the end of forelimb and hindlimb grip strength was assessed to evaluate neuromuscular integrity.
Task 2: Continuous breeding phase F0-generation (1 wk + 14 weeks + Holding period)
Control group and three dose groups. Dose levels were set so that the highest dose was expected to cause decreased nerve function halfway through the task. The middle dose was selected to produced little or no systemic toxicity, whereas the low dose was designed to be a no-effect level.
The animals were housed as breeding pairs for 98 days (continous breeding phase), following seven days of premating exposure to Methacrylamide while singly housed. Endpoints for Task 2 were clinical signs, parental body weight, fertility (number producing a litter/number of breeding pairs), litters per pair, live pups per litter, proprtion of pups born alive, sex of live pups, pup body weights within 24 hours of birth, feed and water consumption, and forelimb and hindlimb grip strength assessments. At the end of the 98 days, the pairs were separated and housed one animal per cage with continued dosing. Any litters born (F1) after the continuous breeding phase were reared by the dam until weaning, and selected weanlings were reared in same-sex groups until 74 +/- 10 days of age. Lactating females were given dosed drinking water at the same dose of Methacrylamide as used during Task 2. Their F1 offspring were used for assessment of second-generation fertility in Task 4 (see below).
Moreover a dominant lethal study was conducted on F0 males.
Task 3 was not conducted (therefore not decribed here), because Task 2 was negative. During the time from day 98 to day 189, time was sufficient to allow for rearing of Task 4 litters. Dosing with assigned concentrations of Methacrylamide continued throughout this period, was discontinued for seven days during week 22 and reinitiated for week 23 to 27. Females were necropsied at day 189. Vaginal cytology was evaluated for the control and high-dose females for 12 days prior to necropsy. At necropsy, females from all dose groups were sacrificed, and body and organ weights were taken.
Males from all dose groups were sacrificed on day 188, but ca. 60 minutes prior to scheduled sacrifice for individual randomly selected males, an endocrine challenge was administered. At sacrifice, cardiac blood was collected, body and organ weights were collected, and an epididymal sperm evaluation was conducted. Selected organs were examined microscopically.
Task 4 Offspring Assessment F1 generation (Control and 1 dosed group)
Assessment of F1 generation, was conducted using offspring from all four dose groups. Animals born after week 15 of Task 2 were weaned, housed two per cage by sex and treatment, and maintained on the same dose of Methacrylamide as their parents until they reached sexual maturity (74 +/- 10 days). 20 control animals of each sex and 20 treated animals of each sex in each treatment group were assigned to Task 4. Animals not selected for Task 4 were euthanized. Task 4 males and females within treatment groups were randomly assigned to breeding pairs, each representing two litters, and housed, one breeding pair per cage. Breeding pairs were cohabited for 7 days or until a vaginal copulatory plug was found, whichever was less, then separated and sigly housed. Dosed water was available ad libitum. After delivery of all of the litters and collection of vaginal smears from females, 12 days prior to being necropsied. Task 4 animals were euthanized and necropsied. Additions to Task 4 were grip strength evaluations and an endocrine challenge test. Data collected included body and selected organ weights, epididymal and testicular spermatozoa evaluations, and concentrations of peripheral serum. Selected organs were examined microscopically after processing.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methacrylamide
EC Number:
201-202-3
EC Name:
Methacrylamide
Cas Number:
79-39-0
Molecular formula:
C4H7NO
IUPAC Name:
methacrylamide
Details on test material:
- Name of test material (as cited in study report): Methacrylamide (MARC) obtained from Pfaltz and Baur, Inc., Waterbury, CT
- Physical state: solid
- Analytical purity: as supplied by producer: >= 99 % relative to a frozen reference standard
- Lot/batch No.: 5524-126-01
- Impurities (identity & concentrations): no data availible by the study report
- Stability under test conditions: no data
- Storage condition of test material: no data

Test animals

Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, NC and Portage, MI)
- Age at study initiation: male: Task 1: 9 weeks, Task 2: 12 weeks
- Weight at study initiation:
- Assigned to test groups randomly: yes, under following basis: stratified randomization based on body weights
- Fasting period before study: no data
- Housing: subsequently housed as breeding pairs or individually
- Diet: pelleted rodent feed, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Task 1, 2 and 4: 22.22 ± 0.01 °C
- Humidity (%): Task 1: Range-finding: 55.2 ± 0.07% ,Task 2: F0-generation: 54.6 ± 0.07%; Task 4: F1-generation: 54.9 ± 0.04%
- Air changes (per hr): no data
- Photoperiod: 14 hours light/ 10 hours dark

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Exposure period: 189 days, 98 days continuous breeding
Premating exposure period (females): premating: 7 days
Duration of test: 27 weeks
Frequency of treatment:
continuously
Doses / concentrations
Remarks:
Doses / Concentrations:
F0: 24, 80 and 240 ppm corresponding to 4.5, 15.4, 49 mg/kg/d||F1: 24, 80 and 240 ppm corresponding to 6.8, 23.8,71.3 mg/kg/d for males and 8-69 mg/kg/d for females
Basis:
nominal in water
No. of animals per sex per dose:
Number of animals (controls): 76: 38 female and 38 male
Number of animals: 36 or 38 per group (18 or 19 males and 18 or 19 females)
Control animals:
yes

Examinations

Statistics:
In Task 1, data were analysed using the Test for Linear Trend, ANOVA, and Tukey's test for pairwise comparison to controls. Most hypotheses in Task 2 and 4 were tested using the nonparametric multiple comparson procedures of Dunn (1964) or Shirley (1977), as modified by Williams (1986). Jonckheere's test (Jonckheere, 1954) was used to ascertain whether there was sufficient evidence of a dose-related response to apply Shirley's test. If the p-value from Jonckheere's test was less than 0.10, Shirley's test was used; otherwise Dunn's test was applied.
For dataxpressed as a proportion, such as fertile/number cohabited, the Cochran- Armitage test (Armitage, 1971) was used to test for dose-related trends, and pairwise comparisons were performed using Chi-squiare (Conover, 1971). Because the number of pups in a litter may influence the average pup weight in that litter, a parametric analysis of covariance (Neter and Wasserman, 1974) was used to test overall equality in average pup weight, after adjustment for average litter size. Pairwise comparison were performed using Dunnett's test.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
240 ppm
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, taking into account the bias caused by diminished body weights of the dose group animals versus control animals the effect of temporarily slightly diminished grip strength described by the study author as slight indication of neurotoxicity in immature animals cannot be confirmed. The invalidity of that finding is aggravated by the lack of body weight data on the test days.
The minor manifestation and reversibility of the described effect is taken into consideration as well.

Consequently, in the absence of any relevant findings on toxicity to reproduction, the NOAEL for developmental toxicity in this study is at the highest administered dose.
The NOAEL (F1) is > 240 ppm (71.3 mg/kg bw/day in males, 69 mg/kg bw/day in females).
Executive summary:

In a one-generation reproduction study (according to modified reproductive assessment continuous breeding protocol (RACB)) Methacrylamide (> 99%) was administered to Swiss CD-1 mice (male/female) in drinking water at dose levels of 0, 24, 80 and 240 ppm (corresponding to F0: 4.5, 15.4, 49 mg/kg bw/day; F1: 6.8, 23.8, 71.3 mg/kg bw/day for males and 8 - 69 mg/kg bw/day for females).

F0: No substance related clinical or histopathological changes. F1: Preweaning growth, survival, food and water consumption was not affected. No treatment related clincal signs, no effect on reproductive competence. Reduced fore- and hindlimb grip strength at 80 and 240 ppm indicates slightly neurotoxic effects. No histopathological  changes. Normal fertility. No dominant lethality.

The observation of a temporarily slightly diminished grip strength in juvenile mice described by the study authors in the one-generation toxicity to reproduction study on methacrylamide (key study) is considered as irrelevant for the NOAEL determination on the following reasons:

 

Published data reveal a strong correlation between body weight of the test animals and grip strength (literature: Maurissen JP et al: Neurotoxicol Teratol. 2003 Sept-Oct; 25 (5): 543-53).

 

The data presented in the one-generation toxicity to reproduction study on methacrylamide show clearly diminished body weights of the dose group animals compared to the control groups at day 21 post partem.

The average difference is 1.04 g for males and 0.89 g for females which is about 8.5 % (males) and 8 % (females) of the body weight at this age.

 Such a depression in body weights in the dose groups as a result of decreased palatability, especially in the beginning of administration, is a common effect which is reported in numerous oral toxicity studies.

 

Moreover, the difference in body weights is expected to be even aggravated as the age of the control animals during the grip strength measurements (average 29.2 days for males and females) was significantly higher than that of dose group animals (average 27.2 days [males and females] for the lowest dose group, 26.8 [females] and 27.3 [males] for the medium dose group and 28.2 [males and females] for the highest dose group).

It is expected that an age increase of 1 - 2 days will lead to significant higher body weights in mice during this phase of strong juvenile growth.

 

In this context it is important to notice that at the day of grip strength measurements, body weights of the animals are not reported. Therefore, the possibility for proof of a direct correlation between body weight and grip strength is lacking in this study.

 

 

In conclusion, taking into account the bias caused by diminished body weights of the dose group animals versus control animals the effect of temporarily slightly diminished grip strength described by the study author as slight indication of neurotoxicity in immature animals cannot be confirmed. The invalidity of that finding is aggravated by the lack of body weight data on the test days.

The minor manifestation and reversibility of the described effect is taken into consideration as well.

 

Consequently, in the absence of any relevant findings on toxicity to reproduction, the NOAEL for developmental toxicity in this study is at the highest administered dose.

The NOAEL (F1) is > 240 ppm (71.3 mg/kg bw/day in males, 69 mg/kg bw/day in females).

 

This study is acceptable and satisfies the requirement for a one-generation reproductive study (modified reproductive assessment continuous breeding protocol (RACB) in Swiss CD-1 mice.