Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Two generation study, RACB design, mice, drinking water (NTP 1992)

NOAEL parenteral/F0 = 240 ppm (49 mg/kg bw/d); no effects observed

NOAEL fertility/F1 = 240 ppm (69/71.3 mg/kg bw/d females/males); no adverse effects observed

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
RACB , Reproductive assessment by continuous breeding
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-01-1990 until 11-11-1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to guideline
Guideline:
other: Continuous breeding study (RACB)
Version / remarks:
Specific study design pulished by Lamb, 1985, Reel et al., 1985
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 (continuous 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, proportion 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 described 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
Specific details on test material used for the study:
- 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
-Chemical characterization and stability determined by RTI
-Reanalysis by MRI
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
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
- Methacrylamide was weighed into 250 ml amber bottles and stored frozen for time of use
- Methacrylamide aliquots were added into 20 l polyethylene caboys with deionized/filtered water (vehicle)-Storage of the solutions
- Analysis were performed periodically to confirm the concentration of methacrylamide in the water
Details on mating procedure:
Task 2 started with a 7-day period of dosing animals housed separately, followed by a 98-day dosing period in which the mice were housed as mating pairs.
Analytical verification of doses or concentrations:
yes
Remarks:
- Analysis were performed periodically to confirm the concentration of methacrylamide in the water
Details on analytical verification of doses or concentrations:
Analytical methods: Thin layer and gas chromatography
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
Dose / conc.:
24 ppm
Remarks:
P1, corresponding to 4.5 mg/kg/d
F1, corresponding to 6.8 mg/kg/d
Dose / conc.:
80 ppm
Remarks:
P1, corresponding to 15.4 mg/kg/d
F1, corresponding to 23.8 mg/kg/d
Dose / conc.:
240 ppm
Remarks:
P1, corresponding to 49 mg/kg/d
F1, corresponding to 71.3 mg/kg/d
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, concurrent vehicle
Details on study design:
In task 1 a range finding study was performed: methacrylamide was tested at levels of 0, 60, l 80, 360, 540, and 720 ppm. Clinical signs consisted of hindlimb splaying in five animals at the top dose. No paralysis was evident, and no other clinical signs were present. Water consumption was increased, but was not related to dose. Body weight gain was not affected by Methylamide exposure. There was no treatment-related change in the number of females carrying vaginal plugs or in the days to plug detection. Female grip strength was decreased slightly only at 710 ppm. Based on these data, concentrations for Task 2 were set at 24. 80, and 240 ppm methacrylamide.
Positive control:
no
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily
Oestrous cyclicity (parental animals):
yes
Sperm parameters (parental animals):
Parameters examined in P, F1 and Task 4 F1 male generations:
Litter observations:
yes
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 data expressed 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)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During the task 2 cohabitation, there were no treatment related differences in body weight.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Female food consumption before and after the cohabitation period was not affected by methacrylamide exposure. Male food consumption was increased at the highest concentration at week 23. At week 16, male food consumption was increased by 10, 12, and 32% compared to controls in the low-, middle-, and high-concentration groups, respectively.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was not affected in either sex by methacrylamide exposure. Based on averaged consumption values (183-204 g water/kg/day), the daily dose estimates for the low-, middle-, and high-concentration groups are 4.5, 15.4, and 49 mg methacrylamide/kg body wt/day.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Methacrylamide exposure was associated with a dose-related increase in male hindlimb grip strength. Some representative data for males and females are presented. Grip strength was tested at weeks 0, 6, 9, 12, and 15 of Task 2, and there was a significant trend to increased hindlimb strength at weeks 6, 12, and 15. Grip strength was not affected in females, and the percentage change in grip strength over the course of the study was not affected by methacrylamide exposure.
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Methacrylamide exposure did not alter estrous cycle length or the time spent in each estrous stage (control length 5.3 ± 5 days).
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
The frequency of abnormal forms was not affected. Epididymal sperm concentration (control value 967 ± 64 X 106/g cauda) was decreased only in the middle-dose group (values from low- to high-dose groups were 800 ± 63, 768 ± 49*,
and 827 ± 73 X 106/g cauda, respectively). Spermatid heads/gram of testis (control 10.l ± 0.5 X 107/g) were decreased significantly only the middle-dose group (low to high dose 8.9 ± 0.6, 8.1 ± 0.5 *, and 8.8 ± 0.6 X 107/g, respectively).
Additionally, only in the high-dose group was epididymal sperm motility lower than controls (60 ± 5% motile for controls, 35* ± 10% motile for the high-dose group).
Reproductive performance:
no effects observed
Description (incidence and severity):
All groups averaged 4.3-5.0 litters/pair during Task 2. MACR did not affect the cumulative days to litter in any group. Neither the number of live pups/litter nor the pup weight was affected by methacrylamide exposure
Exposure of the dams to methacrylamide during lactation had no adverse effect on pup survival to postnatal Day 21 or on body weights before pnd 21.
Key result
Dose descriptor:
NOAEL
Remarks:
parenteral toxicity
Effect level:
49 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No substance related clinical or histopathological changes
Remarks on result:
other: corresponding to 240 ppm
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 male body weights at day 21 in the low-, middle-, and high-dose groups were 8, 5, and 7% lower than controls, respectively. Female body weights in these groups at Day 21 were 7, 6, and 7% less than controls.
At the Task 4 cohabitation at pnd 74 ± 10, the body weights of all groups of females were statistically equivalent, while the body weights of the dosed male groups were reduced by 5, 5, and 6% in the low-, middle-, and high-dose groups. respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food and water consumption, measured the week after cohabitation, was not affected by exposure. Food and water values for control males were 210 ± 13 g food/kg/day and 232 ± 9 g water/kg/day. Food and water consumption
values for control females were 233 ± 12 g food/kg/day and 290 ± 10 g water/kg/day. Using consumption values for both sexes of treated mice, daily doses of methacrylamide can be estimated at ca. 6.8, 23.8, and 71.3 mg methacrylamide/kg/day.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Food and water consumption, measured the week after cohabitation, was not affected by exposure. Food and water values for control males were 210 ± 13 g food/kg/day and 232 ± 9 g water/kg/day. Food and water consumption
values for control females were 233 ± 12 g food/kg/day and 290 ± 10 g water/kg/day. Using consumption values for both sexes of treated mice, daily doses of methacrylamide can be estimated at ca. 6.8, 23.8, and 71.3 mg methacrylamide/kg/day.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
no effects observed
Description (incidence and severity):
There were no changes in epididymal sperm concentration, percentage motility, frequency of abnormal forms, or testicular spermatid head counts (not shown). The length of the estrous cycle inthe 240-ppm group was not different from the controls (control length 4.7 ± 0.1 days).
The fertility and reproductive performance of Task 4 mice were not affected by exposure to 24, 80, or 240 ppm methacrylamide during growth. All groups bad equivalent numbers of live pups/litter, proportion of pups born alive, live pup weight,
adjusted live pup weight, and postpartum dam weight (not shown).
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no differences among the groups in absolute or relative weights of liver, kidneys/adrenals, ovary, prostate, right cauda epididymis, or right testis. There was a
trend to increased relative weight of serninal vesicles, which reached significance in the rniddle-concentration group (at 121 % of control value), but was not statistically different in the low- or high-concentration groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Grossly visible lesions (inflamrnations, uterine hydrometra) were not related to exposure level of methacrylamide
Histopathological findings:
no effects observed
Description (incidence and severity):
Histologie examination of selected tissues from Task 4 mice found no treatment-related effects. There were no visible changes in the nerves at the light-microscope level; there was one case of mild testicular degeneration in each of the
control and the low dose groups, and hydronephrosis in one male from each group, and two in the high-dose group.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
F1 mouse grip strength was significantly affected by methacrylamide only during Week 3 ofTask 4, in the weanling mice.
Male forelimb grip strength was 15, 26, and 29% lower than controls in the low-, middle-, and high-concentration groups, respectively, while male hindlimb grip strength was
19, 12, and 31 % lower than controls. In the females at Week 3, forelimb strength was not affected, but hindlimb strength was reduced by 28, 19, and 32%. These differences were gone by Week 5, and there were no subsequent treatment-related changes in grip strength in Task 4.
In the F1 generation, there were significant reductions in body weight and grip strength in the treated animals at weaning. The changes in body weight were less than 10%.
The grip strength effects were > 10%, but bad disappeared by 5 weeks of life and were not apparent at the time of mating in Task 4. This raises the possibility of an effect on lactational support of the growing pups. Similar to the Fa
mice, there was no observable toxicity in any measured endpoint in Task 4.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Generation:
F1
Effect level:
69 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no effects observed
Remarks on result:
other: corresponding to 240 ppm
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Generation:
F1
Effect level:
71.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no effects observed
Remarks on result:
other: corresponding to 240 ppm
Dose descriptor:
LOEL
Remarks:
neurotoxicity
Generation:
F1
Effect level:
6.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
neuropathology
Remarks on result:
other: corresponding to 24 ppm; not considered as adverse effect as the effect was observed only at PNW3 when the pup body weight was decreased. At PNW5, the grip strength was normal in all dosage groups.
Critical effects observed:
no
Reproductive effects observed:
no

Grip Strength of F0 Mice Exposed to Methacrylamide (Task 2)

ppm Methacrylamide

 

0

24

80

240

Male

 

 

 

 

Forelimb

 

 

 

 

Week 9

109.2±6.3 (10)"

108.6±3.0 (10)

103.0 ::: 2.4 (10)

101.7±5.0 (10)

Week 15

98.8±4.8

101.9 :::: 4.8

98.9 ::: 5.9

103.2±5.5

Hindlimb

 

 

 

 

Week 9

139.7±5.5

148.6±4.5

136.4 ::: 4.9

147.7±3.7

Week 15

132.1:!:5.0

139.8±5.5

138.2::: 5.9

148.8±8.1*t

Female

 

 

 

 

Forelimb

Week 9

105.3:!:3.5 (9)

109.2 :::: 3.4 (10)

101.2 ::: 2.1 (10)

103.1±2.6 (10)

Week 15

96.8 :: 3.4

103.3:::5.4

89.8 ::: 2.5

101.2±4.1

Hindlimb

 

 

 

 

Week 9

141.1 ::: 4.8

152.0::: 5.6

144.5±5.8

145.8±3.0

Week 15

143.8 ::: 8.0

151.2±4.7

144.8:!:4.6

138.8±8.0

" Data arex±SEM(n).

*Significantly different from controls(p<0.05).

tDose-related trend(p<0.05).
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 two-generation toxicity to reproduction in the third postnatal week on methacrylamide is not considered as adverse as the effects disappeared after 5 weeks and thus it is considered as irrelevant for the NOAEL.
Executive summary:

In a two-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).

P0: No substance related clinical or histopathological changes. F1: Preweaning growth, survival, food and water consumption was not affected. No treatment related clinical signs, no effect on reproductive competence.

The observation of the temporarily slightly diminished grip strength in juvenile mice described by the RACB study authors is considered as not adverse and thus as irrelevant for the NOAEL determination,

see discussion in the executive summary on developmental toxicity.

No histopathological changes. Normal fertility. No dominant lethality.

The NOAEL (P0) 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). 

For temporary neurotoxic effects in F1 pups presence of temporary reduced pub body weights on PND21, a LOEL of 24 ppm is defined

 

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

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
49 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
24
Species:
mouse
Quality of whole database:
One screening study acc. OECD 421 with GLP is available and one modified reproductive assessment continuous breeding protocol (RACB) with GLP. The data quality is succificient for assessment.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a two-generation reproduction study according to modified reproductive assessment continuous breeding protocol (RACB; NTP 1992) Methacrylamide 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).


P0: No substance related clinical or histopathological changes. F1: Preweaning growth, survival, food and water consumption was not affected. No treatment related clinical 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 not considered as adverse and thus considered as irrelevant for the NOAEL determination.


 


No histopathological changes. Normal fertility. No dominant lethality.


The NOAEL (P0) 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). 

Effects on developmental toxicity

Description of key information

Mouse, oral gavage, US NTP standards (NTP 1991)


NOAEL maternal toxicity: 60 mg/kg bw/d (increased liver weight)


NOAEL developmental toxicity: 180 mg/kg bw/d (no effects observed)


NOAEL fetal toxicity: 60 mg/kg bw/d (decreased fetal body weight)


 


Rabbit, oral gavage, OECD 414 (CRL 2022)


NOAEL maternal toxicity: 100 mg/kg bw/d (mortality)


NOAEL developmental toxicity: 150 mg/kg bw/d (no effects observed)


 


Rat, oral gavage, OECD 426 (CRL 2021)


NOAEL maternal toxicity: 50 mg/kg bw/d (clinical signs, body weight, food consumption)


NOAEL developmental neurotoxicity: 50 mg/kg bw/d (startle response)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 Feb 2022 to 16 May 2022.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France).
- Age at study initiation: Approximately 17-19 weeks
- Weight at study initiation: Approximately 3000 to 4300 g
- Housing: Animals will be individually housed
- Diet (e.g. ad libitum): KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from
Granovit AG, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): Municipal tap water, Freely available to each animal via water bottles.
- Acclimation period: at least 5 days prior to the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 21°C
- Humidity (%): 40 to 70%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test item will be mortared, vehicle will be added, formulation will be vortexed and subsequently stirred for at least 45 minutes.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
Untreated females will be mated at the Supplier and will be at Day 1 or 2 post-coitum on
arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).
Duration of treatment / exposure:
Day 7 to Day 28 post-coitum
Frequency of treatment:
Once daily
Duration of test:
22 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on results of a Dose Range Finding Study of Meracryl
MAAmide by Oral Gavage in Pregnant New Zealand White Rabbits, Test Facility Study No.
20299410 and in an attempt to produce graded responses to the test item. The high-dose level
should produce some toxic effects, but not excessive lethality that would prevent meaningful
evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The
low-dose level should produce no observable indications of toxicity.



Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 3, 7, 9, 12, 15, 18,
21, 24, 27 and 29 post-coitum.



POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 post-coitum
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and females
with early delivery) will be subjected to an external, thoracic and abdominal examination,
with special attention being paid to the reproductive organs.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Statistics:
Parametric/Non-Parametric: [Body weight, Body weight gain, Food consumption, Gravid Uterine Weight and Gravid Uterus Adjusted Body Weights, Litter Observations]
Levene’s test will be used to assess the homogeneity of group variances.
The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not
significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis
test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s
or Dunn’s test, respectively.
Non-Parametric: [Ovarian and Uterine Examinations, Litter % of Fetuses with Gross/External/Visceral/Skeletal Abnormalities]
The groups will be compared using an overall Kruskal-Wallis test. If the overall
Kruskal-Wallis test is found to be significant, then the above pairwise comparisons will be
conducted using Dunn’s test.
Indices:
Pregnancy Rate (%)
Male Fetuses (%)
Female Fetuses (%)
Pre-Implantation Loss (%)
Post-Implantation Loss (%)
Litter % of Fetuses with Abnormalities


Historical control data:
available
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Erected fur was recorded at 50 and 150 mg/kg/day, at which four females each showed this
sign between post-coitum Days 8-25 and 12-16, respectively. Although this symptom
occurred without a clear dose-response and at a low incidence, it was considered related to
treatment with the test material.
At 150 mg/kg/day, a thin appearance was noted for three females on Day 12 or 16 postcoitum.
Other clinical signs noted did not show a dose-response relationship and at the incidence
observed, these were considered to be unrelated to treatment with the test material.
Mortality:
mortality observed, treatment-related
Description (incidence):
Substance related mortality at dose 150 (mg/kg/day):
Female Nos. 70 and 74 (150 mg/kg/day) were sacrificed in extremis on Day 15 and 16 postcoitum,
respectively, as food consumption was (nearly) absent for 7 or 8 consecutive days and
a body weight loss of 7% was noted for both animals between Days 7 and 12 or 7 and 15
post-coitum, respectively. For Female No. 74, this was accompanied by erected fur on
Days 14-16 post-coitum and a thin appearance on Day 16 post-coitum. At necropsy, relevant
macroscopic findings were a small thymus and less adipose tissue of the whole body for
Female No. 74. Both females were pregnant, with eight live embryos and two late resorptions
for Female No. 70 and nine live embryos for Female No. 74. These sacrifices were considered
related to treatment with the test material. It should be noted however that for Female No. 70
a lower food intake along with some degree of weight loss was already apparent before
treatment was initiated on Day 7 post-coitum. It can therefore not be excluded that this may
have resulted in an increased sensitivity to the effects of treatment for this animal.
Female Nos. 1 (control) and 43 (50 mg/kg/day) were sacrificed in extremis on Day 18 postcoitum
and Day 15 post-coitum, respectively, as blood was noted on the gavage tube directly
after dosing together with laboured breathing. At necropsy, a gavage-related incident was
confirmed for both animals based on the presence of a perforation in the right caudal lobe of
the lung (No. 1) or trachea (No. 43) and frothy red content in the trachea for both animals
(other necropsy findings included lungs that failed to collapse and watery cysts on the oviduct
for Female No. 1 and multifocal foci on the lung and ectopic splenic tissue for Female
No. 43). Therefore, these sacrifices were not related to treatment with the test material. Both
females were pregnant, with thirteen and ten live embryos for Female Nos. 1 and 43,
respectively.
Female No. 2 (control) was sacrificed in extremis on Day 10 post-coitum (pregnant; thirteen
live embryos and one early resorption) based on a (nearly) absent food consumption for 7
consecutive days (from arrival at the test facility onwards) and Female No. 19 (control) was
sacrificed in extremis on Day 25 post-coitum as it started to deliver its offspring. As these
early sacrifices occurred in the control group, they were not related to treatment with the test
material.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg/day, body weight loss (up to 5%) was noted for most animals between Days 7
and 15 post-coitum. This resulted in an overall lower mean body weight gain over Days 7 to
29 post-coitum (255 grams vs 389 grams in the control group). Also, the mean gravid uterus
corrected weight gain was lower (-235 grams vs -116 grams in the control group).

At 100 mg/kg/day, reduced mean body weight gain was noted between Days 7 and 15 postcoitum
(not statistically significant between Days 7 to 9 post-coitum), which resulted in an
overall lower mean body weight gain over Days 7 to 29 post-coitum (245 grams vs 389 grams
in the control group). Also, the mean gravid uterus corrected weight gain was lower (-254
grams vs -116 grams in the control group).

Body weights, body weight gain and gravid uterus adjusted weight gain at 50 mg/kg/day was
considered unaffected by treatment with the test material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 100 and 150 mg/kg/day, a lower mean food consumption was noted from Day 9 and 7
post-coitum, respectively, until Day 21 post-coitum. This resulted in an overall mean food
consumption during the Treatment Period that was 14% and 20% lower compared to control
means, respectively.

Food consumption at 50 mg/kg/day was considered unaffected by treatment with the test
material.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
Uterus gravid weight: see in Body-weight
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations of surviving animals did not reveal any alterations that were
considered to be related to treatment with the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No abortion was reported.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
pre- and post-implantation loss did not show a dose-related trend and remained in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Total litter loss resorption did not show a dose-related trend and remained in the range of normal biological variation.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Early and late resorption did not show a dose-related trend and remained in the range of normal biological variation.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were reported.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No changes in pregnancy duration were reported.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Three control females (Nos. 4, 9 and 21), four females at 50 mg/kg/day (Nos. 24, 30, 32 and
41), one female at 100 mg/kg/day (No. 56) and three females at 150 mg/kg/day (Nos. 77, 86
and 87) were not pregnant. An additional three control females (Nos. 1, 2 and 19), one female
at 50 mg/kg/day (No. 43) and two females at 150 mg/kg/day (Nos. 70 and 74) were sacrificed
in extremis (for details, see Section 7.2.1 (Mortality)), resulting in 16, 17, 21 and 17 females
with live fetuses in the control, 50, 100 and 150 mg/kg/day groups, respectively. These
numbers of females with live fetuses were considered sufficient for adequate evaluation of the
study data.
Other effects:
no effects observed
Description (incidence and severity):
The variation in mean numbers of corpora lutea and implantation sites did not show a dose-related trend and remained in the range of normal biological variation.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
other:
Remarks on result:
other: NOAEL for maternal toxicity =100 mg/kg/day
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal body weights at 50 mg/kg/day were considered not affected by treatment with the test
material.
At 100 and 150 mg/kg/day, mean fetal body weight of both sexes combined was 5.7% and
5.8% lower than control means (not statistically significant), respectively.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no test material-related effects on litter size.
Mean litter sizes were 9.1, 8.9, 9.5 and 9.2 fetuses/litter for the control, 50, 100 and
150 mg/kg/day groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment with the test material.
Mean sex ratios (males:females) were 53:47, 47:53, 44:56, and 56:44 for the control, 50, 100
and 150 mg/kg/day groups, respectively.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
See fetal body-weight changes and reduction in number of live offspring
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test material-related external malformations and variations were recorded. External variations were not observed in this study.
One fetus from the high-dose group (No. 73-L1; 150 mg/kg/day) was observed with omphalocele and based on its single occurrence and as this also occurred for a late resorption in the control group this was considered not to be related to treatment with the test material.
In addition, three late resorptions (10-L3 and 13-L2 of the control group and 80-R11 at 150 mg/kg/day) were observed with a variety of malformations.
The malformations affecting paws at 150 mg/kg/day were also present in the control group
and were therefore considered not related to treatment with the test material.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
No test material-related skeletal variations were noted at 50 mg/kg/day, and no test material related
skeletal malformations were noted at any dose level.
At 100 and 150 mg/kg/day, a higher incidence of thoracolumbar full supernumerary ribs with misaligned ilium was observed but considered to have no significant biological effect.
At 100 and 150 mg/kg/day, a higher mean incidence of thoracolumbar full supernumerary ribs was observed when compared to the control group (mean fetal incidence of 72 and 70%, respectively, vs 35% in the control group). These incidences also exceeded the historical control maximum value and were therefore considered to be related to treatment with the test material.
This skeletal variation was accompanied by a higher incidence of fetuses with misaligned ilium (group fetal incidence of 8% at 100 and 150 mg/kg/day vs 4% in the control group). Although mean litter incidences of misaligned ilium were not statistically significantly increased compared to controls, means were nearly twice the historical control maximum value. Therefore, the increased occurrence of misaligned ilium was also attributed to treatment with the test material at 100 and 150 mg/kg/day.
Also, at 100 and 150 mg/kg/day, the incidence of fetuses with signs of delayed ossification
was higher and consisted of higher incidences of unossified talus (100 and 150 mg/kg/day;
group fetal incidence of 1 and 3%, respectively, vs 1% in the control group) and unossified
hindpaw phalanges (150 mg/kg/day; group fetal incidence of 8%, respectively, vs 2% in the
control group) that were also at or above the historical control maximum value. These signs
of delayed ossification were ascribed to the lower fetal weights rather than being a direct
effect of treatment with the test material.
All other skeletal variations occurred in the absence of a dose-related incidence trend and/or
infrequently. Therefore, they were considered not related to treatment with the test material.
Skeletal malformations were observed in 2 (2), 0 (0), 3 (3) and 1 (1) fetuses (litters) of the control 50, 100, 150 mg/kg/day groups, respectively. The affected structures included femur, rib, sternebra, thoracic vertebra and centre. In groups receiving test material, the malformations were being scored infrequently without a dose response. Therefore, all cases were considered not to be associated to treatment with the test material.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related visceral malformations and variations were recorded.
Two control fetuses (13-R7 and 18-R11) and four fetuses at 100 mg/kg/day (46-L4, 53-L3,
60-L3 and 64-R4) were observed with one or more visceral malformations. These
malformations affected the ventricular septum of the heart, aortic arch, pulmonary trunk,
subclavian artery and kidneys. The findings occurred in one or two fetuses only and in the
absence of a dose-related incidence. All but one finding (pulmonary trunk atresia for one
fetus at 100 mg/kg/day) were within the range of historical control data, and these findings
were noted without a dose-response. All malformations were therefore considered not to be
related to treatment with the test material.
Visceral variations and incidental findings were observed in a range of visceral structures
across all groups. The lower mean number of supernumerary spleen recorded at 100
mg/kg/day was ascribed to a relatively high control mean compared to historical control data.
Therefore, a relationship to treatment with the test material was ruled out.
In all other cases, these variations and incidental findings were either scored infrequently, in
instances comparable to the control group or in the absence of a dose-relationship. Therefore,
they were considered not to be associated to treatment with the test material.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: NOAEL for developmental toxicity = 150 mg/kg/day
Dose descriptor:
NOAEL
Remarks:
Fetal Toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: NOAEL for fetal toxicity = 150 mg/kg/day
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
In a prenatal developmental toxicity study according to OECD TG 414 and GLP principles, the maternal No Observed Adverse Effect Levels (NOAELs) for Methacrylamide in time-mated
female New Zealand White rabbits was established as being 100 mg/kg/day, based on test
material-related mortalities at 150 mg/kg/day. The developmental NOAEL for Methacrylamide was at least 150 mg/kg/day.
Executive summary:

This study was performed according to OECD 414 guideline and followed the GLP principles. 


The objectives of this study were to determine the potential of Methacrylamide to induce
developmental toxicity after maternal exposure during the critical period of organogenesis
and to characterize maternal toxicity at the exposure levels tested when given orally by
gavage to time-mated female New Zealand White rabbits from Day 7 to 28 post-coitum,
inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal
toxicity and developmental toxicity were evaluated.
The dose levels in this study were selected to be 0, 50, 100 and 150 mg/kg/day, based on the
results of the Dose Range Finder Study (Test Facility Study No. 20299410).


Chemical analyses of formulations were conducted once during the study to assess accuracy
and homogeneity. These confirmed that formulations of test material in Water (Elix) were
prepared accurately and homogenously.
The following parameters and end points were evaluated in this study for the F0-generation:
mortality/moribundity, clinical signs, body weights, food consumption, macroscopic
examination, and uterine contents (including corpora lutea, implantation sites, pre- and
postimplantation loss and number of live and dead fetuses).
In addition, the following parameters were determined for the F1-generation: fetal body
weights, sex ratio, external, visceral and skeletal malformations and developmental variations.
At 150 mg/kg/day, two females were sacrificed on Day 15 or 16 post-coitum, based on a
(nearly) absent food consumption for 7 or 8 consecutive days and a body weight loss of 7%
for both animals. One female was also noted with erected fur and a thin appearance before
sacrifice. These sacrifices were considered to be related to treatment with the test material and
therefore adverse.
Additionally, four females were sacrificed in extremis which were considered not related to
treatment with the test material. Two females (one each in the control and 50 mg/kg/day
group) were sacrificed due to a gavage-related incident. Additionally, one female (control)
was sacrificed due to a (nearly) absent food consumption for 7 consecutive days and one
other female (control) was sacrificed as it started to deliver its offspring on Day 25 postcoitum.
At 100 and 150 mg/kg/day, non-adverse reduced food consumption and body weight gain or
weight loss were noted during the initial two weeks of treatment, accompanied by incidental
occurrences of thin appearance and erected fur for individual animals at 150 mg/kg/day. A
non-adverse occurrence of erected fur was also noted at 50 mg/kg/day. Additionally, nonadverse
lower fetal body weights with delayed ossification were noted at these dose levels, as
well as a non-adverse higher incidence of supernumerary ribs with misaligned ilium.
No test material-related changes were noted in any of the remaining maternal parameters
investigated in this study (i.e., macroscopic evaluation, corpora lutea, uterine contents
including implantation sites and pre- and post-implantation loss).
No test material-related changes were noted in any of the remaining developmental
parameters investigated in this study (i.e., litter size, sex ratio, external and visceral
malformations and developmental variations).
In conclusion, based on the results of this prenatal developmental toxicity study in time-mated
female New Zealand White rabbits the following No Observed Adverse Effect Levels
(NOAELs) for Methacrylamide were established:
Maternal NOAEL: 100 mg/kg/day, based on test material-related mortalities at
150 mg/kg/day.
Developmental NOAEL: At least 150 mg/kg/day

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 Mar 1990 - 4 Jun 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
NTP study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
NTP study
GLP compliance:
yes
Remarks:
FDA, 1988
Limit test:
no
Specific details on test material used for the study:
- Supplier: Pfaltz & Baur Inc., Stamford
- Purity: 99 % (GC)
- RTI Lot-number: 5524-126-02
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, North Carolina, USA
- Age at study initiation: no data
- Weight at study initiation: maternal body weights ranged from 25.50 - 32.49 g on gd 0 while mean body weights/dose group ranged from
27.98 - 28.51 g
- Fasting period before study:
- Housing: individually housed in solid-bottom polycarbonate cages with stainless steel wire lids (Laboratory Products, Rochelle Park, NJ) and
Ab-Sorb-Dri cage litter (Laboratory Products, Garfield, NJ)
- Diet (e.g. ad libitum): Purina Certified Rodent Chow ad libitum
- Water (e.g. ad libitum): deionized filtered water ad libitum throughout gestation
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.22°C (original value: 72°F)
- Humidity (%): approximately 55%
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
water
Details on mating procedure:
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
6th - 17th day of gestation
Frequency of treatment:
daily
Duration of test:
17 days
Dose / conc.:
30 mg/kg bw/day
Remarks:
After evaluation of replicate I data, the 30 mg/kg/day dose group was eliminated, since both the 30 and 60 mg/kg/day dose groups caused no effect
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
120 mg/kg bw/day
Dose / conc.:
180 mg/kg bw/day
No. of animals per sex per dose:
15-30 per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily for clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: on the mornings of gd 0, 3, 6, 9, 12, 15, and 17

FOOD AND WATER CONSUMPTION: Yes
- Food and water consumption: on the mornings of gd 0, 3, 6, 9, 12, 15, and 17

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 17
- Organs examined: The maternal body, liver, and intact uterus were weighted and corpora lutea were counted. Uteri which had no visible
implantation sites were stained with ammonium sulfide (10%) to detect very early resorptions (Salewski, 1964)
Fetal examinations:
Live fetuses were weighted, examined for external morphological abnomalities and dissected for visceral examination by a fresh tissue dissection technique (Staples, 1974); Stuckhardt and Poppe, 1984). Half of the fetuses were decapitated prior to dissection; the heads were examined by a free-hand sectioning technique (Wilson, 1965). All fetal carcasses were examined for skeletal malformations (Marr et al., 1988).
Statistics:
General Linear Models (GLM) procedures were applied for the analyses of variance (ANOVA) of maternal and fetal parameters (SAS Institute, 1985). Prior to GLM-ANOVA analysis, an arcsine-square root transformation was performed on all litter-derivived percentage data to normalize the means (Snedecor and Cochran, 1967) and Bartlett's test fot homogenicity of variance was performed on all data to be analyzed by ANOVA (Winer, 1962).GLM-ANOVA analysis determined the significance of dose-response relationships and the significance of dose effects, replicate effects and dose x replicate interactions.
Historical control data:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Effects observed in maternal animals during and after exposure to 0, 60, 120, or 180 mg/kg/day of methacrylamide included swollen eye, swollen tail, rough coat, weight lass, and vaginal bleeding. The swollen eye and one instance of vaginal bleeding occurred in the control group, whereas the swollen tail was due to the tattooing procedure, and thus were not treatment-related
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Lethality occurred at 0 (1 animal), 120 (1 animal), and 180 (1 animal) mg/kg/day from indeterminant causes.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Weight loss after the onset of dosing occurred in 1-2 animals in the control group (gd 7, 8, and 9), 1-3 animals in the 120 mg/kg/day group (gd 7, 8, and 17), and 1-2 animals in the 180 mg/kg/day dose groups (gd 7,8, 9, 10, 14, and 16), and thus seemed slightly more persistent in the high dose group.
Maternal body weight on gd 17(before and after euthanasia), and maternal weight gain during treatment ·and gestation were significantly depressed in the high dose group, compared to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Maternal food consumption was calculated for the intervals of gd 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 17, 6 to 17, and 0 to 17. Relative (g/kg body weight/day) food intake did not differ noticeably between dosed groups and the controls, exhibiting only an increasing trend on gd 12 to 15
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Maternal water consumption was calculated for the intervals of gd 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 17, 6 to 17, and 0 to 17. Maternal water consumption was unaffected by treatment.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At necropsy on gd 17, animals from the 180 mg/kg/day methacrylamide-exposed group had significantly reduced gravid uterine weight; relative maternal liver weight (% body weight) exhibited a dose-related increase, and was significantly increased at both the 120 and 180 mg/kg/day dose levels, although absolute liver weight was not affected.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Effects observed in maternal animals during and after exposure to 0, 60, 120, or 180 mg/kg/day of MAC included swollen eye, swollen tail, rough coat, weight lass, and va~inal b]eeding. The swollen
eye and one instance of vaginal bleeding occurred in the control group, whereas the swollen tail was due to the tattooing procedure, and thus were not treatment-related.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No signs of neurotoxicity were observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
All of the pregnant animals in the 0, 60, and 120 mg/kg/day methacrylamide-treated groups had one or more live fetuses, whereas only 89% (25/28) of the confinned-pregnant animals in the 180 mg/kg/day group had one or more live fetuses.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The percent nonlive implants per litter exhibited a dose-related increasing trend, with the high dose group significantly increased over the control group
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
In the high-dose group, 3 dams had totally resorbed litters (9%) while there were none in the other dose groups or control.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
The percent of litters with resorptions (Ctrl 37%, 60 mg 40%, 120 mg 48% and 180 mg 50%) , or nonlive implants (late fetal deaths plus implants; Ctrl 38%, 60 mg 43%, 120 mg 48% and 180 mg 61%) exhibited dose-related increasing trends.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy on gd 17, 93% (27), 100% (30), 93% (27), and 97% (28) of the mated animals in the control through high-dose groups were confirmed to be pregnant by uterine examination.
Other effects:
no effects observed
Description (incidence and severity):
no effects observed on
- av. number of corpora lutea/dam
- number of impantation sites/litter
- percent preimplantation loss/litter
- percent resorptions/litter
- percent late fetal deaths/litter
- percent of litters with late fetal death
- number of live fetuses/ live litters
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Analysis of fetal body weight data showed the existence of a significant decreasing trend for mean fetal weight (male, female, or both) per litter, with both the 120 and 180 mg/kg/day dose groups significantly below the control group by approximately 7 and 15%, respectively
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
At 180 mg/kg bw/d, increased postimplantation death per litter was observed.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Analysis of fetal body weight data showed the existence of a significant decreasing trend for mean fetal weight (male, female, or both) per litter, with both the 120 and 180 mg/kg/day dose groups significantly below the control group by approximately 7 and 15%, respectively
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
Although there were no statistically significant effects from MAC exposure, the percent of malformed fetuses per litter was noticeably higher than expected in all groups, including the vehicle control group (i.e., 16-21% malformed/ litter as compared to 2.9% malformed litter in 234 historical control litters)
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Effect level:
180 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed
Dose descriptor:
NOAEL
Remarks:
fetotoxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
120 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Results:
Symptoms: at 30 - 60 mg/kg/d: no adverse effects.
          at 120 mg/kg/d: slight maternal effects, and clear evidence of fetal toxicity observed as a decrease in mean fetal body 
          weight per litter.
          at 180 mg/kg/d: mild maternal effects, observed as an increase in relative liver weight, and clear evidence of fetal toxicity, 
          observed as an increased proportion of dead implants per litter, and decreased mean fetal body weight per litter; no external, visceral 
          and skeletal malformations of the fetuses.
Conclusions:
In this developmental toxicity study acc. OECD 414 in mice, the NOAEL for fetal toxicity was considered to be 60 mg/kg/day because mean fetal body weight was reduced.
Executive summary:

In a developmental toxicity study following US standards (NTP method), Methacrylamide (99%) was administered to female Swiss CD-1 mice/dose in deionised water by gavage at dose levels of 0, 60, 120, and 180 mg/kg bw/day from days 6 through 17 of gestation. The study was designed in a two-replicate design with 15-26 animals per dose group per replicate, resulting in 30 animals per dose group for Methacrylamide. 


Maternal toxicity:


No treatment-related maternal mortality was observed.


180 mg/kg/day:


Maternal body weight on GD17, maternal weight gain during treatment and gestation, and corrected maternal weight gain was decreased


Gravid uterine weight was decreased


120 mg/kg/day:


Relative maternal liver weight was increased at 120 mg/kg/day and higher;


60 mg/kg/d:


The maternal NOAEL is 60 mg/kg bw/day. 


Fetal toxicity:


180 mg/kg/day:


At 180 mg/kg/day, increased postimplantation death per litter and decrease in mean fetal body weight (-15%) were observed.


120 mg/kg/day:


Significant, but weak mean fetal body weight was observed at 120 mg/kg/day (-7%).


60 mg/kg/d


The NOAEL for fetal toxicity is 60 mg/kg bw/day.



Teratogenicity:


No effects observed.



The NOAEL for teratogenicity is 180 mg/kg bw/day.




Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
mouse
Quality of whole database:
The quality of the database is high as fully reliable studies from a non-rodent species and rodent species are available. For rodents, supportive data is provided by a Developmental Neurotoxicity Study in rats according to OECD 426.
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

In a study following US standards, pregnant female mice (Swiss CD-1) were dosed daily by gavage with 60, 120 and 180 mg/kg/day of Methacrylamide from GD (Gestational days) 6 to GD17 (NTP 1991). The study was designed in a two-replicate design with 15-26 animals per dose group per replicate, resulting in 30 animals per dose group for Methacrylamide. All animals were killed on GD17 and examined for maternal body weight, implant status, fetal weight, sex and morphological development.


Maternal toxicity was observed at 180 mg/kg/day as decreased maternal body weight on GD17, decreased maternal weight gain during treatment and gestation, and decreased gravid uterine and relative liver weight. At 120 mg/kg/day, relative maternal liver weight was increased. At 60 mg/kg/day, no effects were observed.


No developmental toxicity was observed at any dose level. 


Fetotoxicity was observed at 180 mg/kg/day as decreased fetal body weights (average and both sexes separately) and increased postimplantation death per litter. At 120 mg/kg/day, decreased fetal body weights (average and both sexes separately) was observed. At 60 mg/kg/day, no effects were observed.


In conclusion, based on the results of this prenatal developmental toxicity study in Swiss CD-1 mice the following No Observed Adverse Effect Levels (NOAELs) for Methacrylamide were established:
Maternal NOAEL: 60 mg/kg/day, based on test material-related liver weight effects at 120 mg/kg/day.
Developmental NOAEL: 180 mg/kg/day, in absence of any effects.


Fetotoxicity NOAEL: 60 mg/kg/day, based on test material-related fetal body weight effects at 120 mg/kg/day.


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In a study according to OECD 414 guideline and the GLP principles time-mated female New Zealand White rabbits were exposed to 50, 100 and 150 mg/kg/day of Methacrylamide by oral gavage from Day 7 to 28 post-coitum (CRL, 2022). 


The No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.
The dose levels in this study were selected based on the results of a Dose Range Finder Study.


The following parameters and end points were evaluated in this study for the F0-generation:
mortality/moribundity, clinical signs, body weights, food consumption, macroscopic examination, and uterine contents (including corpora lutea, implantation sites, pre- and postimplantation loss and number of live and dead fetuses).
In addition, the following parameters were determined for the F1-generation: fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.
At 150 mg/kg/day, two females were sacrificed on Day 15 or 16 post-coitum, based on a (nearly) absent food consumption for 7 or 8 consecutive days and a body weight loss of 7% for both animals. One female was also noted with erected fur and a thin appearance before sacrifice. These sacrifices were considered to be related to treatment with the test material and therefore adverse.
Additionally, four females were sacrificed in extremis which were considered not related to treatment with the test material. Two females (one each in the control and 50 mg/kg/day group) were sacrificed due to a gavage-related incident. Additionally, one female (control) was sacrificed due to a (nearly) absent food consumption for 7 consecutive days and one other female (control) was sacrificed as it started to deliver its offspring on Day 25 postcoitum.
At 100 and 150 mg/kg/day, non-adverse reduced food consumption and body weight gain or weight loss were noted during the initial two weeks of treatment, accompanied by incidental occurrences of thin appearance and erected fur for individual animals at 150 mg/kg/day. A non-adverse occurrence of erected fur was also noted at 50 mg/kg/day. Additionally, nonadverse lower fetal body weights with delayed ossification were noted at these dose levels, as
well as a non-adverse higher incidence of supernumerary ribs with misaligned ilium.
No test material-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e., macroscopic evaluation, corpora lutea, uterine contents including implantation sites and pre- and post-implantation loss).
No test material-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e., litter size, sex ratio, external and visceral malformations and developmental variations).
In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female New Zealand White rabbits the following No Observed Adverse Effect Levels (NOAELs) for Methacrylamide were established:
Maternal NOAEL: 100 mg/kg/day, based on test material-related mortalities at 150 mg/kg/day.
Developmental NOAEL: At least 150 mg/kg/day


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In a developmental neurotoxicity study according to OECD TG 426 and GLP principles, Methacrylamide was given by gavage to 25 female Wistar Han rats per dose from Day 6 post-coitum up to and including lactation Day 20 in doses of 15, 50 and 150 mg/kg/day to determine the potential functional and/or morphological effects of the developing nervous system of the offspring that may arise from exposure in utero and during lactation/ early life (CRL 2021).


For the F0-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption and gross necropsy findings.


For the F1-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, functional observations including acoustic startle response, learning and memory test (Biel maze), motor activity and grip strength, gross necropsy findings, brain weights and histopathologic examinations of central and peripheral nervous tissues (including brain morphometry).


In addition, the following reproduction/developmental parameters were determined for the F0-generation: gestation duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex and macroscopy).


In the F0-generation, no test item-related mortality occurred during the study period. Arena observation parameters as determined on post-coitum Days 8 and 17 and lactation Days 7 and 14 were considered not affected by treatment with the test item. At 150 mg/kg/day, adverse clinical signs (piloerection, hunched posture, uncoordinated movements and abnormal gait of the hindlegs/ forelegs) were recorded. In addition, at 150 mg/kg/day, a treatment-related weight loss was recorded between post-coitum Day 6 and 9 for most animals, ranging from -1 to -15%. Mean body weight at this dose level remained lower throughout treatment (0.90x and 0.91x of the control mean at the end of the post-coitum and lactation period, respectively). Body weight gain at 150 mg/kg/day was also lower during the post-coitum period (0.66x of the control mean at the end of the post-coitum period). These changes in body weight were accompanied by lower food intake throughout most of the post-coitum and lactation period (0.82x and 0.89x of control at the end of the lactation period for absolute and relative food intake, respectively). Based on their magnitude and duration of change, these body weight and food intake changes were considered adverse. Necropsy revealed red foci on the thymus for several surviving animals at 150 mg/kg/day. Although no histopathology was conducted on the thymus, the observed incidence suggested that this finding was related to treatment with the test item. At 15 and 50 mg/kg/day, no treatment related effects were observed.


No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. duration of gestation, parturition, sex ratio, maternal care, litter size, live birth index, viability index, weaning index and macroscopy).


In the lactation phase of the F1-generation, mortality incidences occurring among pups during lactation were considered not related to treatment. A lean appearance was recorded for most pups of 14/22 litters between PND 17 and 20 at 150 mg/kg/day, which was in line with lower mean pup body weights at this dose level recorded throughout the pre-weaning period (0.77x of controls for males and females combined on PND 21). No treatment-related clinical signs and no treatment-related changes in body weight were recorded for pups at 15 and 50 mg/kg/day.


In the post-weaning phase of the F1-generation at 150 mg/kg/day, lower mean body weights were recorded for both sexes throughout the study period (0.89x and 0.93x of controls for males and females, respectively, at the end of the study period). Body weight gain during the post weaning phase was higher for males and females throughout the study period (statistically significant on all occasions; 1.25x and 1.29x of control for males and females, respectively, at the end of the study period), indicating that body weights showed a tendency towards recovery. Accordingly, food consumption of males dosed with 150 mg/kg/day was lower throughout the study period and statistically significant on most occasions (mean of means for absolute food consumption was 0.91x of control), while absolute food consumption of females at 150 mg/kg/day was statistically significantly lower in Weeks 1 to 3 of the post weaning period only.


At 15 and 50 mg/kg/day, body weight gain (absolute and relative) and food intake (before and after correction for body weight) were considered not affected by treatment with the test item.


No test item-related changes were noted in any of the other F1-generation post-weaning developmental parameters investigated in this study (i.e mortality, clinical signs, balanopreputial separation, vaginal opening and macroscopy).


Regarding neurotoxicity parameters in the F1-generation, treatment-related effects consisted of lower grip strength on PND 20, 35 and 60, reduced startle response on PND 25 and 60 and reduced motor activity on PND 13 at 150 mg/kg/day. At 50 mg/kg/day, treatment related effects were confined to a slightly lower startle response on PND 25.


Essentially reversible effects were recorded for motor activity and grip strength at 150 mg/kg/day, based on which these effects were considered not adverse. Reduced startle response at 150 mg/kg/day was not completely reversible during the study period. While there were no supportive histopathological correlates and means remained within historical control ranges, the magnitude and non-reversible nature of the reduced startle response was considered to represent an adverse effect.


In conclusion, based on the results of this Developmental Neurotoxicity Study, the following No Observed Adverse Effect Levels (NOAEL) of Methacrylamide were established:


Maternal NOAEL:   50 mg/kg/day (based on clinical signs, weight loss or reduced weight gain and reduced food intake at 150 mg/kg/day)


Developmental Neurotoxicity NOAEL:     50 mg/kg/day (based on non-complete recovery and magnitude of reduced startle response at 150 mg/kg/day)

Justification for classification or non-classification

There is no indication of effects on fertility which would be relevant for classification for the respective hazard. Thus, a classification on reproductive toxicity is not warranted.


There is no indication of relevant effects on developmental toxicity in studies with mice and rabbits which would be relevant for classification for the respective hazard. Effects on Developmental Neurotoxicity in the rat (OECD 426) and mouse (RACB) were either reversible and seen in presence of maternal toxicity (grip strength, mouse & rat) or well within historical control levels (startle response, rat). In all those cases, these effects were considered as biologically not relevant. Thus, a classification as developmental toxicant is considered as not warranted.

Additional information