Methacrylamide

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Principles of method if other than guideline:

Remark regarding OECD 414 in rabbits:
The selected additional endocrine disrupter relevant endpoints (AGD in fetuses and thyroid hormones in dams) were included in TG 414 following a feasibility study addressing scientific and technical concerns regarding inclusion of additional endpoints in the test method (2). The 2018 update is to include rat-specific requirements in the TG 414; thus applies to rats and not to rabbits.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methacrylamide

EC Number:

201-202-3

EC Name:

Methacrylamide

Cas Number:

79-39-0

Molecular formula:

C4H7NO

IUPAC Name:

methacrylamide

Test material form:

solid

Details on test material:

Batch (Lot) Number: 11110320

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (supplier) : Röhm GmbH, Darmstadt, Germany
-Batch No.: 11110320
- Purity: 99.88%
-Expiry Date: 31 March 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In freezer (≤ -15°C)
In freezer (≤ -15°C)
Test Facility Test Item Number: 210604/B
Purity/Composition Correction
Factor:
No correction factor required
Test Item Handling: No specific handling conditions required
Stability in Water: Stability for at least 24 hours at room temperature under normal laboratory light conditions, for at least 8 days in the refrigerator and for at least 3 weeks in the freezer ( -15°C) is confirmed over the concentration range 1 to 100 mg/mL (solutions), Project 20215967

Test animals

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).
- Number of Females: 88 (time-mated).
- Number of Fetuses Expected: Approximately 792 fetuses (88 litters x 9 fetuses).
- Age at study initiation: Approximately 17-19 weeks
- Weight at study initiation: Approximately 3000 to 4300 g

ENVIRONMENTAL CONDITIONS
- 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.
- 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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Water (Elix, Millipore S.A.S., Molsheim, France) Specific Gravity: 1.0

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.
Frequency of preparation: At least weakly
Storage conditions set to maintain: 4 °C

STABILITY ANALYSIS:
Stability analyses performed previously in conjunction with the method development and
validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The objective of the analytical study was to determine the accuracy of preparation and
homogeneity of Methacylamide in formulations.
Accuracy
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in
agreement with target concentrations (i.e., mean sample concentration results were within or
equal to 90-110% of target concentration).
No test material was detected in the Group 1 formulation.
Homogeneity
The formulations of Group 2 and Group 4 were homogeneous (i.e., coefficient of variation
≤ 10%).

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 4

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:

RANGE FINDING STUDY / 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 .
The range finding study was performed with 6 groups of 6 pregnant rabbits (each dose group at) dose concentrations of 0, 70, 100, 130, 200 and 300 mg/kg bw/day by oral application. Groups 5 and 6 were added to the study based on the results of Groups 1-4.
All females of the 200 and 300 mg/kg bw/d dose groups were sacrificed for ethical reasons between days 14 and 16 post-coitum, based on body weight loss and/or absent or severely reduced food consumption. Dosing of these animals was discontinued from day 14 post-coitum onwards.

In the 200 mg/kg bw/d range finding group, erected fur was noted for 3/6 females between days 11 and 15 post-coitum, body weight loss (up to -10%) was recorded for all females between days 7 and 12 post-coitum. Body weight gain was observed for most animals after dosing was discontinued on day 14 post-coitum. Due to the early termination, gravid uterine weights could not be determined. Reduced to absent food consumption for 5/6 females from start of treatment onwards and from day 11 post-coitum for 1/6 females. Following cessation of dosing on Day 14 post-coitum, food consumption recovered between days 14 and 16 post-coitum. Macroscopic observations at necropsy did not reveal any alterations that were considered to be related to treatment with the test material. All females were pregnant. Number of corpora lutea and
implantation sites was normal. Fetal Findings were not determined due to preterminal sacrifice between day 14 and 16 post-coitum.

In the 300 mg/kg bw/d range finding group, erected fur was noted for 5/6 females between Days 9 and 15 post-coitum. Body weight loss (up to -11%) was recorded for all females between days 7 and 12 post-coitum. Body weight gain was observed for most animals after dosing was discontinued on day 14 post-coitum. Due to the early termination, gravid uterine weights could not be determined. Reduced to absent food consumption for 6/6 females from start of treatment onwards. Following cessation of dosing on day 14 postcoitum, food consumption recovered for most animals between days 14 and 16 post-coitum. One female showed watery content in the cecum which was also distended with gas. This finding is not commonly encountered for rabbits of this age and strain. Therefore, although this finding occurred in a single female only, it could not be excluded that these were related to treatment with the test material. Except for one female, all females were pregnant. Number of corpora lutea and implantation sites was normal. Fetal Findings were not determined due to preterminal sacrifice between day 14 and 16 post-coitum.


Normal body weight gain (before and after correction for gravid uterus weight) or slight body weight gain was observed in the 70, 100 and 130 mg/kg bw dose groups. In each one of the 3 lower dose groups one animal was sacrificed due to observations (details see Attachment: Result table of range finding study 20299410). No or marginal clinical sign were noted in the lower dose groups. Lower food consumption from day 7 post-coitum onwards, which was most prominent between Days 12 and 18 post-coitum and additionally between day 27 and 29 post-coitum in the 130 mg/kg bw /d dose group. Overall mean food consumption over days 7 and 29 post-coitum was 5% (70 mg/kg bw/d), 3 % (100 mg/kg bw/d) and 14 % (130 mg/kg bw/D) lower compared to control.
In the 3 lower dose groups, macroscopic observations at necropsy did not reveal any alterations that were considered to be related to treatment with the test material. All females were pregnant. Number of corpora lutea and implantation sites was normal. Mean values for pre- and postimplantation loss were considered not affected by treatment with the test material. Litter sizes and fetal body weights were normal. External examination of the fetuses did not show any abnormalities.
No effects have been observed in the control group.
For details see Attachment: Result table of range finding study 20299410) in Overall remarks, Attachments

Based on the minor/moderate findings in the 70, 100 and 130 mg/kg bw/day dose groups, but severe toxicity observed at 200 and 300 mg/kg bw/day, doses for the main study were selected at 50, 100 and 150 mg/kg bw/day.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Dermal irritation (if dermal study):

not examined

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

Water consumption and compound intake (if drinking water study):

not specified

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

1

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

2

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

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Summary of Maternal Performance and Mortality
Sex: Female	0 mg/kg/day		50 mg/kg/day	100 mg/kg/day	150 mg/kg/day
	Group 1		Group 2	Group 3	Group 4
Day(s) Relative to Mating (Litter: A)
Group Size - Females		22	22	22	22
Number of Females Pregnant [f]	N+ve	19	18	21	19
	%	86.4	81.8	95.5	86.4
Female with Live Fetuses [f]	N+ve	18	18	21	19
	%	94.7	100.0	100.0	100.0
Total Resorptions [f]	N+ve	1	0	0	0
	%	5.3	0.0	0.0	0.0
Female with all Nonviable [f]	N+ve	1	0	0	0
	%	5.3	0.0	0.0	0.0
Terminal Euthanasia [f]	N+ve	19	21	22	20
	%	86.4	95.5	100.0	90.9
Unscheduled Death/Euthanasia [f]	N+ve	3	1	0	2
	%	13.6	4.5	0.0	9.1
Unscheduled Euthanasia [f]	N+ve	2	1	0	2
	%	9.1	4.5	0.0	9.1
Delivered [f]	N+ve	1	0	0	0
	%	4.5	0.0	0.0	0.0

 

 

 

Summary of Ovarian and Uterine Examinations and Litter Observations I
Sex: Female	0 mg/kg/day		50 mg/kg/day	100 mg/kg/day	150 mg/kg/day
	Group 1		Group 2	Group 3	Group 4
Day(s) Relative to Mating (Litter: A)
Female with Live Fetuses	N+ve	16	17	21	17
	%	100.0	100.0	100.0	100.0
Number of Corpora Lutea [k]	Mean	11.4	10.1	10.7	10.5
	SD	1.9	2.2	1.6	1.9
	N	16	17	21	17
	%Diff	-	-12.1	-6.3	-7.9
Number of Implantations [k]	Mean	9.9	8.9	10.0	9.7
	SD	2.9	2.0	1.8	1.4
	N	16	17	21	17
	%Diff	-	-9.5	1.3	-1.7
Pre-implantation Loss (%) [k]	P	12.87	10.36	6.44	6.89
	SD	21.40	12.19	11.12	8.40
	N	16	17	21	17
	%Diff	-	-19.47	-49.94	-46.46
Total Number of Fetuses [k]	Mean	9.1	8.9	9.5	9.2
	SD	2.7	2.0	1.9	1.6
	N	16	17	21	17
	%Diff	-	-2.7	3.8	0.6
Number of Live Fetuses [k]	Mean	9.0	8.9	9.5	9.2
	SD	2.7	2.0	1.9	1.6
	N	16	17	21	17
	%Diff	-	-1.3	5.3	2.0
Number of Dead Fetuses [k]	Mean	0.1	0.0	0.0	0.0
	SD	0.3	0.0	0.0	0.0
	N	16	17	21	17
	%Diff	-	-100.0	-100.0	-100.0
Number of Early Resorptions [k]	Mean	0.5	0.0	0.5	0.5
	SD	0.7	0.0	0.7	1.0
	N	16	17	21	17
	%Diff	-	-100.0	4.8	-5.9

 

 

Summary of Ovarian and Uterine Examinations and Litter Observations II
Sex: Female	0 mg/kg/day		50 mg/kg/day	100 mg/kg/day	150 mg/kg/day
	Group 1		Group 2	Group 3	Group 4
Day(s) Relative to Mating (Litter: A)
Number of Late Resorptions [k]	Mean	0.3	0.1	0.0	0.1
	SD	0.6	0.2	0.0	0.2
	N	16	17	21	17
	%Diff	-	-76.5	-100.0	-76.5
Total Number of Resorptions [k]	Mean	0.8	0.1	0.5	0.5
	SD	1.1	0.2	0.7	1.0
	N	16	17	21	17
	%Diff	-	-92.2	-30.2	-29.4
Post-implantation Loss (%) [k]	Mean	8.18	0.65	5.33	5.35
	SD	11.73	2.69	7.01	10.56
	N	16	17	21	17
	%Diff	-	-92.01	-34.85	-34.55
Number of Live Male Fetuses [k]	Mean	4.9	4.2	4.3	5.1
	SD	2.5	1.7	2.1	1.8
	N	16	17	21	17
	%Diff	-	-14.3	-12.1	5.0
Number of Live Female Fetuses [k]	Mean	4.1	4.7	5.2	4.1
	SD	2.0	1.9	1.6	1.9
	N	16	17	21	17
	%Diff	-	14.1	25.8	-1.6
Live Male Fetus/Litter (%) [k]	Mean	53.07	47.20	43.74	56.39
	SD	18.62	15.62	18.37	17.77
	N	16	17	21	17
	%Diff	-	-11.06	-17.58	6.26
Live Female Fetuses/Litter (%) [k]	Mean	46.93	52.80	56.26	43.61
	SD	18.62	15.62	18.37	17.77
	N	16	17	21	17
	%Diff	-	12.51	19.89	-7.08
Mean Fetal Weight males (g) [G]	Mean	39.84	40.81	37.20	37.47
	SD	5.91	6.25	3.77	4.79
	N	16	17	20	17
	%Diff	-	2.44	-6.62	-5.96

 

 

 

Summary of Ovarian and Uterine Examinations and Litter Observations III
Sex: Female	0 mg/kg/day		50 mg/kg/day	100 mg/kg/day	150 mg/kg/day
	Group 1		Group 2	Group 3	Group 4
Day(s) Relative to Mating (Litter: A)
Mean Fetal Weight females (g) [G]	Mean	39.69	40.11	37.36	37.24
	SD	6.50	4.97	4.32	5.80
	N	16	17	21	17
	%Diff	-	1.06	-5.87	-6.18
Mean Fetal Weight all (g) [G]	Mean	39.83	40.54	37.55	37.52
	SD	4.92	5.41	3.97	4.90
	N	16	17	21	17
	%Diff	-	1.79	-5.73	-5.80

 

 

Summary of Fetal abnormalities Findings
		0 mg/kg/day group 1	50 mg/kg/day group 2	100 mg/kg/day group 3	150 mg/kg/day group 4
	Number of fetuses examined	144	151	199	156
	Number of litters evaluated	150	152	199	157
	Number of fetuses examined	16	17	21	17
	Number of litters evaluated	16	17	21	17
Trunk, Omphalocele - Malformnations	Fetuses N (%) Litters N(%)	0(0.00) 0(0.0)	0(0.00) 0(0.0)	0(0.00) 0(0.0)	1(0.65) 1(5.9)

 

Applicant's summary and conclusion

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:

In a developmental toxicity study acc. OECD 414 Methacrylamide (99.88 %) was administered by gavage to time-mated female New Zealand White rabbits at doses of 0, 50, 100 and 150 mg/kg bw/day in water, from day 7 to 28 post-coitum.

 

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.

Reduced food consumption was recorded at 100 and 150 mg/kg/day during the first two weeks of treatment, resulting in a 14% and 20% lower food consumption over the treatment period compared to control, respectively. This was accompanied by weight loss and/or reduced weight gain during the first week of treatment and lower mean gravid uterus corrected weight gain at both 100 and 150 mg/kg/day. These changes in food intake and body weight were accompanied by incidental occurrences of thin appearance and erected fur at 150 mg/kg/day. Erected fur was also incidentally noted at the low dose (50 mg/kg/day) and possibly represented a test material-related effect. As all of these findings were generally reversible as treatment progressed, they were considered not adverse.

 

Test material-related lower fetal body weights (6% lower than controls) were noted at both 100 and 150 mg/kg/day, which were likely related to the reduced food consumption and weight gain of the dams as described above. Based on the small magnitude of the change, this was considered not adverse.

 

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