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Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
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 referenz 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
Sex:
male/female
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
Details on study schedule:
Number of generation studies: 1
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.
Reproductive indices:
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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
> 240 ppm
Sex:
male/female
Basis for effect level:
other: No substance related clinical or histopathological changes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings:
no effects observed

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
reproduction
Generation:
F1
Effect level:
> 240 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Results: Methacrylamide induced no changes in fertility or pup end points at any dose in F0 animals.
There were no treatment-related differences in body weights. Grip strength was variably changed 
and significantly (12%) increased in males at week 15. Consistent with the lack of reproductive 
effects in Task 2, Methacrylamide produced no change in the dominant lethal test after Task 2. 
In the absence of detectable effects on fertility end points, Task 3 (crossover mating to determine 
the affected sex) was not conducted. At necropsy, sperm concentration was reduced by 21% at 80 ppm, 
and sperm motility was reduced by approximately 42% at 240 ppm.In the F1 animals, there was slight 
neurotoxicity at postnatal day 21, measured as decreases in forelimb grip strength in males (26 - 29%) 
and hindlimb grip strength (12 - 31%). These decrements in grip strength were not apparent by week 5,
nor were they seen again as the animals matured. At cohabition at approximately postnatal day 74, 
male body weights were significantly reduced approximately 5 % in all treated groups. 
In F1 matingtrial, there were no treatment-related effects on any reproductive end point, nor were 
any necropsy end points meaningfully altered.
No dominant lethality.

Applicant's summary and conclusion

Conclusions:
F0 Swiss mice, exposed to Methacrylamide in drinking water at dose levels as high as 240 ppm for 27 weeks, had normal fertility with no evidence
of dominant lethality or reproductive or neurotoxicity.
The observation of the 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.
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.

The observation of the 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, discussion see at developmental toxicity.

No histopathological  changes. Normal fertility. No dominant lethality.

The NOAEL (F0) is > 240 ppm (49 mg/kg bw/day)

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.