Registration Dossier

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 assesement.
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)) 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 study authors in the one-generation toxicity to reproduction study on methacrylamide (key study) is considered as considered as adverse and thus as irrelevant for the NOAEL determination, see discussion under "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). 

Effects on developmental toxicity

Description of key information

Study according to US NTP standards, mouse, oral gavage (NTP 1991)

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

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

NOAEL teratogenicity 180 mg/kg bw/d (no effects observed)

A developmental toxicity study with rabbits as 2nd, non-rodent species is planned based an an ECHA CCH request.

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

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

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

Link to relevant study records
Reference
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 rnaternal animals during and after exposure to 0, 60, 120, or 180 rmg/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 treatrnent-related
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Lethality occurred at 0 (1 animal), 120 (1 anirmal), 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
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Maternalwater 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.
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.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No signs of neurotoxicity were observed
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 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:
fetotoxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
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
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, 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 (according to OECD 414) Methacrylamide (99%) was administered to female Swiss CD-1 mice/dose in deionised water by gavage at dose levels of 0, 30, 60, 120, and 180 mg/kg bw/day from days 6 through 17 of gestation.

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 moderate as data from a non-rodent species are currently missing. The generation of supportive data for rodents is curently ongoing by performance of 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 NTP standards, pregnant female (Swiss CD-1) mice were dosed daily by gavage with 60, 120 and 180 mg/kg/day of Methacrylamide from GD (Gestational days) 6 to GD17. All animals were killed on GD17 and examined for maternal body weight, implant status, fetal weight, sex and morphological development.

No treatment-related maternal mortality was observed. Maternal body weight on GD17, maternal weight gain during treatment and gestation, and corrected maternal weight gain was decreased at 180 mg/kg/day. Relative maternal liver weight was increased at 120 mg/kg/day and higher; gravid uterine weight was decreased at 180 mg/kg/day.

The maternal NOAEL was considered to be 60 mg/kg/day.

Significant but weak mean fetal body weight reduction was observed at 120 mg/kg/day (-7%). At 180 mg/kg/day, increased postimplantation death per litter and decrease in mean fetal body weight (-14%) were observed. Morphological development was not affected.

The NOAELs for fetal toxicity and teratogenicity were considered to be 60 mg/kg/day and 180 mg/kg/day, respectively.

The slight reduction in mean fetal body weight was not confirmed in the RACB study in the same mouse strain in doses up to 49 mg/kg bw/d (see details above).

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. The main argument here is the reversibility of the effect as the grip strength was normal at the 5th post natal week.

The second argument is the coincidence of the appearance of the grip strength effect together with reduced pup body weigth. 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; for rats; mice are expected to show comparable but not identical patterns).

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.

A third argument is the slightly different age of the animals at girp strength testing: the aforemenioned 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.

Justification for classification or non-classification

There is no indication of reproductive or developmental toxicity which would be relevant for classification for thr respective hazard. Thus, a classification is not warranted.

Additional information