Allyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproduction/Developmental Toxicity Screening Test (rat, oral, gavage, OECD 422) with Allyl methacrylate: NOAEL for reproductive performance (mating and fertility): 60 mg/kg bw/day, the highest dose tested.

Two-Generation Reproduction Toxicity Study (rat, oral, gavage), Read across: Methyl Methacrylate: NOAEL general, systemic toxicity: 50 mg/kg bw/day (due to adverse effects on food consumption), NOAEL for fertility and reproductive performance (P and F1 parental animals): 400 mg/kg bw/day (according to OECD 416)

Two-Generation Reproduction Toxicity Study (rat, oral, gavage), Read across: Acrolein which is the most relevant toxic metabolite of Allyl methacrylate. NOAEL general, systemic toxicity: 1 mg/kg bw/day due to stomach lesions and diminished body weight gain. NOAEL for fertility and reproductive performance (P and F1 parental animals): 6 mg/kg bw/day.

Converted to allyl methacrylate a NOAEL of 13.5 mg allyl methacrylate/kg bw/d was calculated based on OECD 416 with acrolein..

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-10-19 to 2004-12-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted on 22 March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA guideline OPPTS 870.3650, July 2000

Deviations:

no

Principles of method if other than guideline:

The study was conducted as a combined repeated dose toxicity study with Reproduction / Developmental Toxicity Screening Test. In this part all parameters concerning Reproduction / Developmental Toxicity were considered.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: males were 8 weeks old and the females were 10 weeks old
- Weight at study initiation: 303 g (range: 280 g to 332 g) for the males and 229 g (range: 199 g to 273 g) for the females
- Fasting period before study: no
- Housing: The animals were housed individually (except during mating) in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust was placed under each cage. Shortly before parturition, the females were moved to polycarbonate cages (43.0 x 21.5 x 20.0 cm) with autoclaved sawdust and wood shavings as nesting material (SICSA, Alfortville, France).
- Diet: The animals had free access to SSNIFF R/M-H pelleted maintenance diet, Batch Nos. 2764132 and 4764439 (SNIFF Spezialdiäten GmbH, Soest, Germany) distributed weekly.
- Water: The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2004-10-12 To: 2004-12-07

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Vehicle
The vehicle was corn oil, Batch No. 103K0107, supplied by Sigma (Saint-Quentin-Fallavier, France).

Dosage formulation preparation
The test item dosage formulations were prepared by suspending Allyl Methacrylate (AMA) in corn oil in order to achieve the concentrations of 0.6, 3 and 12 mg/mL and were stored at +4°C or up to 9 days protected from light.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: during the 2 week mating period, one male and one female from the same dose group were co-habitated during the night until pregnancy occurs or the mating period is over
- Proof of pregnancy: vaginal plug / sperm in vaginal smear

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of the dosage formulations
Homogeneity
Two dosage formulations were prepared at 0.2 and 15 mg/mL of AMA (in corn oil) in order to cover all the concentrations intended for use in this study. From both dosage formulations, duplicate samples were taken at three different levels of the container (top, middle, bottom) and analyzed for concentration of the test item just after preparation (day 0) and after 9 days of storage at +4°C (protected from light).
Stability
The two dosage formulations prepared at 0.2 and 15 mg/mL of AMA in corn oil for homogeneity analysis were sampled after 0 (just after preparation), 4 and 9 days storage at +4°C (protected from light). The aliquots (n = 2) sampled on day 4 were stored frozen at -20°C pending analysis on the last sampling occasion, when all samples were assayed. The mean concentrations measured on days 0 and 9 for homogeneity were taken as the initial and final values for the stability test.
Concentration
The concentration of samples taken from each dosage formulation (including the control) prepared for use in weeks 1, 2, 4 and 7 was determined.

Results
A satisfactory agreement was observed between the nominal and actual concentrations of the test item in the dosage formulations analyzed since the deviations from nominal concentration remained in an acceptable range of ± 10%. Furthermore, there was a satisfactory correspondence between the nominal and the measured concentrations of the test item in the vehicle.

Duration of treatment / exposure:

Males: 15 days before mating, through mating and post-mating periods through sacrifice (minimum 4 weeks);
Females: 15 days before mating, through mating period, pregnancy and lactation, until day 5 post-partum inclusive (or until sacrifice, for unmated females);

Frequency of treatment:

once daily

Details on study schedule:

Each animal was given the appropriate dosage formulation once a day, at approximately the same time each day, 7 days a week, according to the following schedule:
in the males:
. 15 days before mating, during the mating and post-mating periods until sacrifice (approximately 5 weeks in total),
in the females:
. 15 days before mating,
. during the mating period,
. during pregnancy and lactation, until day 5 post-partum inclusive (or until sacrifice, for un-mated females).
Day 1 corresponds to the first day of treatment period.

Remarks:

Doses / Concentrations:
3, 15 and 60 mg Allyl Methacrylate/kg/day
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected following the results of a previously conducted study CIT/Study No. 28198 TSR in which rats were dosed (gavage) withAllyl Methacrylate (AMA), Batch. 00216X, at 0, 25, 75 and 150 mg/kg/day for 10 consecutive days. In this study, 150 mg/kg/day resulted in morbidity and mortality, both sexes in the 25 and 75 mg/kg/day groups showed signs of hypersalivation during the dosing period, males showed signs of piloerection at 75 mg/kg/day, and the females in the 75 mg/kg/day group had increased liver weight.
Taking into account that the duration of the dosing period for this study was nearly 6 weeks in females, the high dose-level was set at 60 mg/kg/day. The intermediate and low dose-levels were selected in order to identify a No Effect Level.

Positive control:

not required

Parental animals: Observations and examinations:

- CLINICAL OBSERVATIONS: Clinical observations were conducted daily throughout the study period.

- DETAILE CLINICAL OBSERVATIONS (DCO): Conducted on all rats pre-exposure and weekly throughout the study. The DCOs included cage-side, handheld and open-field observations that were recorded categorically or using explicitly defined scales.

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded weekly throughout the study. Female body weights were performed weekly for the premating and mating periods. Maternal body weights were recorded on gd 0, 7, 14, 17 and 20. Females that delivered were weighed on ld 1 and 5. Females that failed to mate or deliver were weighed on a weekly basis for the study duration.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined weekly for males and females during the pre-mating period. Due to co-housing, food consumption was not measured during mating. During gestation, food consumption for the females was measured on gd 0, 7, 10, 14, 17 and 20. After parturition, food consumption was measured on ld 1 and 5.

OTHER:
Females were observed for signs of parturition beginning on gd 20. The first day the presence of a litter was noted was designated ld 1.

Litter observations:

All litters were examined as soon as possible after delivery and the following information was recorded: litter size, the number of live and dead pups and the sex. Pup clinical observations and body weights were recorded on pnd 1 and 5. In addition, any physical abnormalities or demeanor changes were recorded during the lactation period.

Postmortem examinations (parental animals):

In five male and five females from each group, the following tissues were harvested and preserved in appropriate materials: macroscopic lesions, adrenals, brain, cecum, colon, duodenum, epididymis, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes, ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids, trachea, urinary bladder, and uterus. An examination of the uterus for the number of implantation sites was recorded. Weights were also recorded for the adrenals, brain, epididymis, heart, kidneys, liver, spleen, testes and thymus.

Postmortem examinations (offspring):

Pups found dead and sacrificed on ld 6 were carefully examined for external gross abnormalities and a macroscopic evaluation was performed. All litters were examined as soon as possible after delivery.

Statistics:

The following sequence was used for the statistical analyses of body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data. If there were 3 or less animals/sex/group remaining, no statistical analysis was performed. If there were 5 or less animals/sex/group remaining for 3 or more groups, the data were analyzed using the Dunn test. If there was 5 or less animals/sex/group remaining for less than 3 groups, the data were analyzed using the Mann-Whitney/Wilcoxon test. If there was 5 or more animals/sex/group, the data were analyzed by Kolmogorov-Lillefors test to test for the normality of the distribution of the data. If it was not normal, the values were logarithmically transformed and retested for normality. If it still did not meet the criteria for normality, the original values were analyzed by the Dunn test. If the data were normal, and there were 3 or more groups, the data were assessed for homogeneity of variance between groups with the Bartlett test. If not homogeneous, the data were analyzed by the Dunn test; if homogeneous, the data were assessed by the Dunnett test. If the data were normal, and there was less than 3 groups, the data were assessed for homogeneity of variance between groups with the Fisher test. If not homogeneous, the data were assessed using the Mann-Whitney/Wilcoxon test; if homogeneous, the data were analyzed by the Student test. The statistical comparison of results of motor activity (horizontal andrearing movements recorded from five males and five females per group) was performed, according to the following steps: step 1: normality and homogeneity of variances (Kolmogorov-Smirnov and Bartlett test respectively), step 2: parametric (ANOVA plus Dunnett test if necessary) or non-parametric (Kruskal-Wallis plus Dunn test if necessary) tests was conducted based on the results obtained at step 1. These statistics were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc).

Reproductive indices:

The following calculations were made:
- Pre-implantation loss
- Post-implantation loss
- Mating index
- Fertility index
- Gestation index

Offspring viability indices:

The following calculations were made:
- Live birth index
- Viability index

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hypersalivation was observed in a dose-related manner in males and females given 15 or 60 mg/kg/day during the premating period (from week 2) and then in females during gestation and lactation. This clinical sign was not considered to be adverse as it likely represents a reaction to the dosing procedure and not a direct effect of AMA toxicity. At 60 mg/kg/day, one female experienced hypotonia and half-closed eyes on day 5 p.p.. There was no disturbance of autonomal or physiological functions at any dose-levels and the motor activity of the animals was considered to be unaffected by treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on group mean male body weight gain during the premating phase and only a marginal reduction in body weight gain was observed at 15 and 60 mg/kg/day (p<0.05) during the mating period. There was no effect of treatment on body weights of females during the premating period, gestation or lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on food consumption for females during the premating period, gestation or lactation.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no changes in hematological parameters that could be considered toxicologically relevant

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For blood biochemistry investigation, one female dosed with 60 mg/kg/day had increased total bilirubin concentration and another one had increased biliary acid concentration, these changes were correlated with histopathological findings in the liver.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no effects of treatment on pH, specific gravity or volume of urine produced by the males in any group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The necropsy of the adult males and females revealed the presence of yellowish areas in the liver of 2/5 females in the 60 mg/kg/day dose group.
At histopathology, several foci of degenerated/necrotic hepatocytes, together with slight periportal fibrosis, slight biliary proliferation, and greenish-pigment-laden macrophages, were noted in 3/5 females given 60 mg/kg/day. There were no other treatment-related histopathological changes for either sex at any dose.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating Data: There was no effect of treatment on mating at any dose-level.
Fertility Data: The male and female fertility indices were unaffected by treatment; all mated females were pregnant with live fetuses.
Delivery Data: The duration of gestation was similar between the control and test item-treated groups and close to the normal value of 21 days. There was no effect of treatment on the mean number of liveborn pups or on pup death after birth. There were no external abnormalities in the control or test item-treated groups.

Hypersalivation was observed in a dose-related manner in males and females given 15 or 60 mg/kg/day during the premating period (from week 2) and then in females during gestation and lactation. This clinical sign was not considered to be adverse as it likely represents a reaction to the dosing procedure and not a direct effect of AMA toxicity. At 60 mg/kg/day, one female experienced hypotonia and half-closed eyes on day 5 p.p.. There was no disturbance of autonomal or physiological functions at any dose-levels and the motor activity of the animals was considered to be unaffected by treatment. There was no effect of treatment on group mean male body weight gain during the premating phase and only a marginal reduction in body weight gain was observed at 15 and 60 mg/kg/day (p<0.05) during the mating period. There was no effect of treatment on body weights of females during the premating period, gestation or lactation. There was no effect of treatment on food consumption for females during the premating period, gestation or lactation. There were no changes in hematological parameters that could be considered toxicologically relevant.
For blood biochemistry investigation, one female dosed with 60 mg/kg/day had increased total bilirubin concentration and another one had increased biliary acid concentration, these changes were correlated with histopathological findings in the liver. There were no effects of treatment on pH, specific gravity or volume of urine produced by the males in any group.
-Mating Data: There was no effect of treatment on mating at any dose-level.
-Fertility Data: The male and female fertility indices were unaffected by treatment; all mated females were pregnant with live fetuses.
-Delivery Data: The duration of gestation was similar between the control and test item-treated groups and close to the normal value of 21 days. There was no effect of treatment on the mean number of liveborn pups or on pup death after birth. There were no external abnormalities in the control or test item-treated groups.
The necropsy of the adult males and females revealed the presence of yellowish areas in the liver of 2/5 females in the 60 mg/kg/day dose group.
At histopathology, several foci of degenerated/necrotic hepatocytes, together with slight periportal fibrosis, slight biliary proliferation, and greenish-pigment-laden macrophages, were noted in 3/5 females given 60 mg/kg/day. There were no other treatment-related histopathological changes for either sex at any dose.

Key result

Dose descriptor:

NOAEL

Remarks:

(mating/fertility)

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed

Critical effects observed:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(but none of these differences attained statistical significance)

Coldness to the touch was noted in one pup in the control group, two pups (one litter) in the 15 mg/kg/day group ( and eight pups (three litters) at 60 mg/kg/day. This coldness to the touch was sporadically observed in control animals (one pup) and the litter incidence in the 15 mg/kg/day group (two litters, two pups) did not indicate a treatment-related effect. At 60 mg/kg/day, based on the incidence (three litters, eight pups) and the duration of these observations (from day 3 p.p. generally), a relationship to treatment with the test item could not be ruled out.
The other findings were considered to be distributed among the groups with no dose-related incidence, and as such, none of them were attributed to the test-treatment.

-Pup Body Weights and Sex Ratio: There was a slight reduction in body weight gain for male and female pups at 15 or 60 mg/kg/day. As none of these differences attained statistical significance, none of them were considered to be adverse signs of toxicity. The sex ratios on days 1 and 5 post-partum were similar in the control and test item-treated groups, and close to a theoretical value of 50 %.

-Pup Necropsy: No relevant findings were observed in the pups sacrificed on day 6 post-partum.

Key result

Dose descriptor:

NOAEL

Remarks:

(developmental toxicity)

Generation:

F1

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed

Critical effects observed:

no

Reproductive effects observed:

not specified

Pup Body Weight and Weight Change:
Sex	Males				Females
Dose (mg/kg/day)	0	3	15	60	0	3	15	60
Body weight (g)
Days 1	7.2	7	6.9	6.9	6.8	6.9	6.6	6.4
Days 5	12	11.6	10.9	10.8	11.3	11.2	10.5	10.2
Body weight change(g)
Days 1-5	4.7	4.6	4	3.9	4.5	4.3	3.9	3.9
		-2%	-15%	-17%		-4%	-13%	-13%

Conclusions:

In this study, the NOAEL for reproductive performance (mating and fertility): 60 mg/kg bw/day, the highest dose tested.

Executive summary:

In this study according to OECD 422 with GLP, Sprague-Dawley rats (10/sex/group) were administered Allyl methacrylate via gavage at 0 (corn oil), 3, 15, or 60 mg/kg bw/day. Males were treated once daily during pre-mating and the mating period for a minimum of 4 weeks. Females were treated once daily during pre-mating, mating and gestation and through post natal day (PND) 5 (the day of pup birth was designated PND 1). Males were sacrificed after the mating period, and females were sacrificed with their litters on post natal day (PND) 6. There were no treatment-related effects on mating, fertility indices, the duration of gestation, mean number of live born pups or on pup death after birth. There were no gross external pup abnormalities in the control or Allyl methacrylate-treated groups. The NOAEL for reproductive performance (mating and fertility) was 60 mg/kg bw/day (the highest dose tested).

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Methacrylates
Alkyl and glycol acrylates and methacrylates have been shown to hydrolyze to acrylic or methacrylic acid and the corresponding alcohol (McCarthy, 1997; cited in the OECD SIDS IUCLID data set of 2009), but the affinity and turnover of this reaction is reduced with increasing chain length but the overall reaction is consistent and reasonably predictable.
In addition, Jones (2002) conducted an elaborate series of in vitro and in vivo studies on carboxylesterase activity with 7 methacrylates ranging from methyl methacrylate to octyl methacrylate (with increasing ester size) for several tissues (blood, liver, skin and nasal epithelium) from rats and humans (cited in the OECD SIDS IUCLID data set of 2009). It was concluded that methacrylate esters are rapidly hydrolyzed by carboxylesterases to methacrylic acid (MAA) and the respective alcohol. Clearance of the parent ester from the body is in the order of minutes.
Based on the results of these studies, allyl methacrylate can be expected to hydrolyze rapidly into allyl alcohol and methyl methacrylate.

Allyl acetate
Allyl acetate can be used for read across as alkyl- and other smaller acetates will also rapidly be hydrolyzed by carboxylesterases so that allyl acetate will be hydrolyzed into the common metabolites allyl alcohol and the ubiquitous acetic acid.

Acrolein
Auerbach SS et al, (2008) summarized as follows: Metabolism studies of allyl acetate and allyl alcohol strongly suggest these chemicals are protoxicants that are metabolized to acrolein, a highly reactive, unsaturated aldehyde. Upon oral administration allyl acetate is rapidly reduced by carboxyl esterases in the stomach, liver and blood to yield allyl alcohol and acetic acid. Allyl alcohol is then further oxidized to acrolein by alcohol dehydrogenase. Acrolein can subsequently be detoxified by aldehyde dehydrogenase to acrylic acid or be conjugated by glutathione.
The glutathione adducts can potentially be converted by cytochromes P450 to the hard electrophile and known mutagen/ carcinogen, glycidaldehyde (Barros et al., 1994; Feron et al., 1991). Alternatively, degradation products of glutathione adducts,S-(3-hydroxypropyl)mercapturic acid andS-(2-carboxyethyl) mercapturic acid, are found in the urine of rats administered acrolein, allyl alcohol, allyl chloride, allyl amine, or allyl bromide (Kaye, 1973; Sanduja et al., 1989). In support of this proposed mechanism of toxicity, pharmacological inhibition of carboxylesterase or alcohol dehydrogenase was shown to attenuate allyl ester mediated hepatotoxicity (Silver and Murphy, 1978). Similar studies with allyl alcohol demonstrated that inhibition of alcohol dehydrogenase leads to a reduction in hepatotoxicity (Reid, 1972). Furthermore,mice hypomorphic for alcohol dehydrogenase are resistant to allyl alcohol induced liver injury (Belinsky et al., 1985). Inhibition of aldehyde dehydrogenase enhances the toxicity of allyl alcohol suggesting that not only does the production of acrolein affect toxicity, but its metabolic breakdown also plays a role in liver injury (Rikans, 1987).

Conclusion
In conclusion available data of allyl alcohol and methyl methacrylate and other alkyl methacrylates can be used for assessment of the toxicokinetic parameters of allyl methacrylate. For methyl methacrylate and allyl alcohol (2 -propen-1 -ol) extensive data are available. These study data strongly support the role of acrolein as the primary reactive species and toxicant following exposure of allyl acetate or allyl alcohol. (see also 7.1 Basic toxicokinetics)


For structural and physico chemical properties of the parent substances,t the read across substances and the metabolites see the attached document

Reason / purpose for cross-reference:

read-across source

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 37 (±1) days at the beginning of treatment; (F1) x wks
- Weight at study initiation: (P) Males: 127.5 - 151.0 g; Females: 110.5 - 145.1 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages (Becker & Co., Castrop-Rauxel, Germany), floor area of about 800 cm², with the following exceptions: 1) During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. 2) Pregnant animals and their litters were housed together until PND 21 (end of lactation).
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water: ad libitum
- Acclimation period: (P) about 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10 or 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals that took into account the analytical results of the stability verification. For the test substance preparation, the specified amount of test substance was weighed into an Erlenmeyer flask, topped up (shortly under the marking) with 1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The Erlenmeyer flask was sealed and the preparation was intensely mixed with a magnetic stirrer. A magnetic stirrer was used to keep the preparations homogeneous during treatment of the animals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (= GD 0)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory ten times during the study period (among other things at the beginning and towards the end) for verification of the concentrations. The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (50 and 400 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.

Duration of treatment / exposure:

until one day before sacrifice

Frequency of treatment:

once daily

Details on study schedule:

- F1 parental animals not mated until 75 days after selected from the F1 litters.
- Selection of parents from F1 generation after weaning (PND 21).
- Age at mating of the mated animals in the study: [...] weeks

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

F0 generation parental animals: 25
F1 generation parental animals: 25

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: first day of the premating period and then once a week at the same time of the day (in the morning) until sacrifice. The following exceptions are notable for the female parental animals: 1) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. 2) Females showing no positive evidence of sperm in vaginal smears were weighed once a week during the mating interval (solely for calculation of dose volume). 3) Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 14 and 21. 4) Females without litter were weighed once a week during the lactation phase (solely for calculation of dose volume).



OTHER:
- Food consumption: In general, food consumption was determined once a week for the male and female F0 and F1 parental animals. After the 10th test week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly for GD 0-7, 7-14 and 14-20. During the lactation period (animals with litter) food consumption was determined for PND 1-4, 4-7, 7-14 and 14-21.

Oestrous cyclicity (parental animals):

Estrous cycle length and normality were evaluated daily for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. The evaluations were continued throughout the mating period until the female exhibited evidence of mating. Moreover, at the scheduled necropsy a vaginal smear was microscopically examined to determine the stage of the estrous cycle for each F0 and F1 female.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight, cauda epididymis weight, prostate weight, seminal vesicles including coagulation glands weight, sperm head count in testis, sperm head count in cauda epididymis, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, clinical symptoms, sexual maturation


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE AND GROSS NECROPSY
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined: Anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, cauda epididymis, prostate, seminal vesicles including coagulation glands, ovaries, uterus, spleen, brain, pituitary gland, thyroid glands (with parathyroid glands). The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively: Vagina, cervix uteri, uterus, ovaries (BOUIN), oviducts, left testis (BOUIN), left epididymis (BOUIN), seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroid glands (with parathyroid glands), all gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

OTHER:
- Differential Ovarian Follicle Count (DOFC) in F1 generation: From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, respectively, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope (magnification: 100x) on each of these slides, – according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press). To prevent multiple counting on serial slides – especially of the growing follicles – only follicles with an oocyte with visible chromatin on the slide were counted. The number of each type of follicle was recorded individually for ovary 1 and ovary 2 of every animal on any of the slide levels (level 1-10), giving in summary the incidence of each type of the follicles by using EXCEL sheets for the reporting of the results. Finally, the results of all types of follicles were summarized for all animals per group in dose groups 10 and 13. As primordial follicles continuously develop into growing follicles, the assessment of the follicles was extended to the combined incidence of primordial plus growing follicles. In general, the fifth slide of the left and right ovary was evaluated for histological findings. Whenever the diagnosis: ”no abnormalities detected” was used for the ovaries, this implicates all functional statuses of follicles, especially corpora lutea, were present. An attempt was made to correlate gross lesions with histopathological findings.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age. All F2 offspring were sacrificed after weaning (~ PND 21).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
Animals with notable findings or abnormalities were further evaluated on a case-by-case basis, depending on the type of finding noted. After the scheduled sacrifice the brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. For the calculation of the relative organ weights, the pup body weights, determined routinely during the in-life phase on PND 21, were used.

Reproductive indices:

- Male reproduction data: The mating partners, the number of mating days until vaginal sperm could be detected in the female, and the gestational status of the female were noted for F0 and F1 breeding pairs. For the males, mating and fertility indices were calculated for F1 and F2 litters according to the following formulas: Male mating index (%) = (number of males with confirmed mating)/(number of males placed with females) x 100. Male fertility index (%) = (number of males proving their fertility)/(number of males placed with females) x 100.
- Female reproduction and delivery data: The mating partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 and F1 females. For the females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to the following formulas:
Female mating index (%) = (number of females mated)/(number of females placed with males) x 100.
Female fertility index (%) = (number of females pregnant)/(number of females mated) x 100.
Gestation index (%) = (number of females with live pups on the day of birth)/(number of females pregnant) x 100.
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 and F2 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth)/(total number of pups born) x 100.
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = (number of implantations – number of pups delivered)/(number of implantations) x 100.

Offspring viability indices:

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21. Furthermore, viability and lactation indices were calculated according to the following formulas:
Viability index (%) = (number of live pups on day 4 (before culling) after birth)/(number of live pups on the day of birth) x 100.
Lactation index (%) = (number of live pups on day 21 after birth)/(number of live pups on day 4 (after culling) after birth) x 100.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test group 03 (400 mg/kg bw/d)
• Statistically significantly decreased food consumption in parental males during premating weeks 5 - 10 (up to 7%)
• Statistically significantly decreased food consumption in parental females during premating weeks 1 - 3 (up to 6%), weeks 5 - 8 (up to 7%) and weeks 9 - 10 (about 6%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 10%)
• Statistically significantly decreased food consumption in parental females during lactation days 4 - 7 (about 7%)

Test group 02 (150 mg/kg bw/d)
• Statistically significantly decreased food consumption in parental females during premating weeks 1 - 2 (about 5%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 7%)

Test group 01 (50 mg/kg bw/d)
• no test substance-related adverse effects/findings

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Under the conditions of the present 2-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 400 mg/kg bw/d for the F0 parental rats, the highest dose tested.
The NOEL (no observed effect level) is 50 mg/kg bw/d for the F0 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.
The NOAEL for fertility and reproductive performance for the F0 parental rats is 400 mg/kg bw/d, the highest dose tested.

Dose descriptor:

NOEL

Remarks:

food consumption

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Dose descriptor:

LOEL

Remarks:

food consumption

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: consequence of reduced appetite observed in the F0 parental females

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

General systemic toxicity

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test group 03 (400 mg/kg bw/d)
Statistically significantly decreased food consumption in parental males during premating weeks 0 - 1 (about 14%) and weeks 8 - 10 (up to 7%)
• Statistically significantly decreased food consumption in parental females during premating weeks 0 - 1 (about 10%) and weeks 9 - 10 (about 8%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 14 (up to 8%)
• Statistically significantly decreased body weights in parental males during weeks 0 - 5 (up to 17%) and weeks 10 - 11 (up to 6%)
• Statistically significantly decreased body weights in parental females during premating weeks 0 - 1 (up to 16%)

Test group 02 (150 mg/kg bw/d)
• no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d)
• no test substance-related adverse effects/findings

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

The NOEL (no observed effect level) is 50 mg/kg bw/d for the F1 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.
The NOAEL for fertility and reproductive performance for the F1 parental rats is 400 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity, in the F1 of the test substance is 400 mg/kg bw/d, the highest dose tested.

Dose descriptor:

NOEL

Remarks:

food consumption

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other:

Remarks:

effects on food consumption beeing a consequence of reduced appetite observed at the LOEL of 150 mg/kg bw/d in the F0 parental females

Dose descriptor:

NOAEL

Remarks:

fertility & reproductive performance

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no dverse effects observed

Critical effects observed:

no

Reproductive effects observed:

no

- Analytics:

The various analyses:

• demonstrated the stability of the test substance preparations over a period of 7 days at room temperature
• confirmed the homogeneous distribution of the test substance in the vehicle (1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop hydrochloric acid)
• showed that the prepared concentrations were close to the expected values ranging between 86.8 and 113.2% of the nominal concentrations

Analytical values (range):

Test group	Nominal Dose (mg/kg bw/d)	Analytical Dose (mg/kg bw/d) [minimum]	Analytical Dose (mg/kg bw/d) [maximum]	% Nominal Dose [minimum]	% Nominal Dose [maximum]
00 / 10	0	0	0
01 / 11	50	43.40	46.61	86.8	93.2
02 / 12	150	132.90	169.80	88.6	113.2
03 / 13	400	359.20	379.90	89.8	95.0

 

Conclusions:

No study on 2-generation reproduction toxicity is available with Allyl methacrylate. Allyl methacrylate will be metabolized in a first step into allyl alcohol and methacrylic acid. In the same way methyl methacrylate will be metabolized rapidly into methacrylic acid and respective alcohol. Therefore the 2 -generation toxicity study in rats with methyl methacrylate will be used for the read across approach of the methacrylic moiety of allyl methacrylate.

The NOAEL for general, systemic toxicity is 400 mg/kg/d for the parental rats in the highest dose tested.
The NOEL 50 mg/kg bw/day for the P parental rats, based on effects on food consumption observed at the LOAEL of 150 mg/kg bw/day in the P parental females.
The NOAEL for fertility and reproductive performance for the P and F1 parental rats is 400 mg/kg bw/day, the highest dose tested.
The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance is 400 mg/kg bw/day, the highest dose tested.

Executive summary:

No study on 2-generation reproduction toxicity is available with Allyl methacrylate. Allyl methacrylate will be metabolized in a first step into allyl alcohol and methacrylic acid. In the same way methyl methacrylate will be metabolized rapidly into methacrylic acid and respective alcohol. Therefore the 2 -generation toxicity study in rats with methyl methacrylate will be used for the read across approach of the methacrylic moiety of allyl methacrylate.

The study was performed according to OECD TG 416 in compliance with GLP. Methyl Methacrylate was administered to groups of 25 male and 25 female healthy young Wistar rats (P parental generation) as an aqueous preparation by stomach tube at dosages of 0; 50; 150 and 400 mg/kg body weight/day. At least 73 days after the beginning of treatment, P animals were mated to produce a litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were treated with the test substance at dosages of 0; 50; 150 and 400 mg/kg body weight/day post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals.

Control parental animals were dosed daily with the vehicle (1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop hydrochloric acid).

The mid- and high-dose parental animals (400 mg/kg bw/d) showed clinical signs of systemic toxicity. The only relevant clinical observation was temporary salivation during a short period after dosing, which is considered to be test substance-induced. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.

In the mid- and high-dose (150 and 400 mg/kg bw/d) P generation animals, dose-related intermittent reductions of food consumption were noted, either during premating, gestation and lactation phases of this study. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group.

High dose F1 parental males had statistically significant lower body weights during several study segments, which led to a statistically significant reduction of the mean terminal body weight resulting in secondary weight changes of brain.

High dose parental females had statistically significant lower body weights during the first weeks after weaning. This weight decrease during major phases of sexual maturation led to an apparent marginal delay of vaginal patency. This minor delay did, however, not result in any corroborative pathological findings nor did it adversly effect F1 female cyclicity, fertility and reproduction. Thus, an influence of the test substance on female sexual maturation is not assumed.

Pathological examinations revealed no test-substance-related changes in organ weights, gross lesions, changes in differential ovarian follicle counts or microscopic findings, apart from an increase in kidney and liver weights in male and female animals in both generations which is presumably related to the treatment. There was no histopathologic lesion observed, that could explain the weight increase. It is regarded to be an adaptive change, most likely caused by an increase in metabolic activity in the two organs, which does not lead to histopathologic findings. It is not regarded to be an adverse effect.

There were no indications from clinical examinations as well as gross and histopathology, that the administration of methyl methacrylate via the diet adversely affected the fertility or reproductive performance of the P or F1 parental animals up to and including a dose of 400 mg/kg bw/day. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups and ranged within the historical control data of the test facility.

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 400 mg/kg bw/day. Up to this dose level, the test substance did not adversely influence pup viability and pup body weights. Sex ratio and sexual maturation was not directly affected at any dose level, inclusive the high-dose group (400 mg/kg bw/day).

Conclusion:

Under the conditions of the present 2-generation reproduction toxicity study the NOAEL(no observed adverse effect level) forgeneral, systemic toxicityis 400 mg/kg bw/d for the parental rats, the highest dose tested.

The NOEL (no observed effect level) is 50 mg/kg bw/d for the F1 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.

The NOAEL for fertility and reproductive performance for the F1 parental rats is 400 mg/kg bw/d, the highest dose tested.

 The NOAEL for developmental toxicity, in the F1 of the test substance is 400 mg/kg bw/d, the highest dose tested.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Reference 3

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1992

Justification for type of information:

Methacrylates
Alkyl and glycol acrylates and methacrylates have been shown to hydrolyze to acrylic or methacrylic acid and the corresponding alcohol (McCarthy, 1997; cited in the OECD SIDS IUCLID data set of 2009), but the affinity and turnover of this reaction is reduced with increasing chain length but the overall reaction is consistent and reasonably predictable.
In addition, Jones (2002) conducted an elaborate series of in vitro and in vivo studies on carboxylesterase activity with 7 methacrylates ranging from methyl methacrylate to octyl methacrylate (with increasing ester size) for several tissues (blood, liver, skin and nasal epithelium) from rats and humans (cited in the OECD SIDS IUCLID data set of 2009). It was concluded that methacrylate esters are rapidly hydrolyzed by carboxylesterases to methacrylic acid (MAA) and the respective alcohol. Clearance of the parent ester from the body is in the order of minutes.
Based on the results of these studies, allyl methacrylate can be expected to hydrolyze rapidly into allyl alcohol and methyl methacrylate.

Allyl acetate
Allyl acetate can be used for read across as alkyl- and other smaller acetates will also rapidly be hydrolyzed by carboxylesterases so that allyl acetate will be hydrolyzed into the common metabolites allyl alcohol and the ubiquitous acetic acid.

Acrolein
Auerbach SS et al, (2008) summarized as follows: Metabolism studies of allyl acetate and allyl alcohol strongly suggest these chemicals are protoxicants that are metabolized to acrolein, a highly reactive, unsaturated aldehyde. Upon oral administration allyl acetate is rapidly reduced by carboxyl esterases in the stomach, liver and blood to yield allyl alcohol and acetic acid. Allyl alcohol is then further oxidized to acrolein by alcohol dehydrogenase. Acrolein can subsequently be detoxified by aldehyde dehydrogenase to acrylic acid or be conjugated by glutathione.
The glutathione adducts can potentially be converted by cytochromes P450 to the hard electrophile and known mutagen/ carcinogen, glycidaldehyde (Barros et al., 1994; Feron et al., 1991). Alternatively, degradation products of glutathione adducts,S-(3-hydroxypropyl)mercapturic acid andS-(2-carboxyethyl) mercapturic acid, are found in the urine of rats administered acrolein, allyl alcohol, allyl chloride, allyl amine, or allyl bromide (Kaye, 1973; Sanduja et al., 1989). In support of this proposed mechanism of toxicity, pharmacological inhibition of carboxylesterase or alcohol dehydrogenase was shown to attenuate allyl ester mediated hepatotoxicity (Silver and Murphy, 1978). Similar studies with allyl alcohol demonstrated that inhibition of alcohol dehydrogenase leads to a reduction in hepatotoxicity (Reid, 1972). Furthermore,mice hypomorphic for alcohol dehydrogenase are resistant to allyl alcohol induced liver injury (Belinsky et al., 1985). Inhibition of aldehyde dehydrogenase enhances the toxicity of allyl alcohol suggesting that not only does the production of acrolein affect toxicity, but its metabolic breakdown also plays a role in liver injury (Rikans, 1987).

Conclusion
In conclusion available data of allyl alcohol and methyl methacrylate and other alkyl methacrylates can be used for assessment of the toxicokinetic parameters of allyl methacrylate. For methyl methacrylate and allyl alcohol (2 -propen-1 -ol) extensive data are available. These study data strongly support the role of acrolein as the primary reactive species and toxicant following exposure of allyl acetate or allyl alcohol. (see also 7.1 Basic toxicokinetics)


For structural and physico chemical properties of the parent substances,t the read across substances and the metabolites see the attached document

Reason / purpose for cross-reference:

read-across source

Species:

rat

Strain:

other: Crl:CD (SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Breeding Labs., Inc.
- Age at study initiation: 58 days
- Weight at study initiation: Males: 278-375 g; Females: 187-251 g
- Housing: 1-2 rats/sex/cage. All adult rats were housed in suspended stainless-steel cages above absorbent paper liners. Cages were changed every other week and cage pan liners were changed three times a week. During the cohabiting period (maximum period of 21 days), male and female rats of the same dosage group were housed together (1:1). No later that day 14 of presumed gestation, female rats were transferred to a nesting box (1 female per nesting box). During the 21-day postpartum period, each dam was housed in a common nesting box. Nesting boxes contained Bed-o'cobs bedding which was changed three times a week.
- Diet (e.g. ad libitum): certified rodent chow 5002 in meal form ad libitum
- Water (e.g. ad libitum): reverse osmosis membrane treated water (with up to 1 ppm chlorine) ad libitum.
- Acclimation period: 2 weeks

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Solutions of Acrolein were prepared daily with deionized water. These were analyzed prior to dosing using a spectrophotometer and found to be within 1% of the desired concentration. The stability of Acrolein in deionized water at concentrations of 0.2 and 1.2 mg/ml were investigated at 6 hours after preparation. A loss of 7% and 4% for the low-dose and high-dose, respectively, was observed

Details on mating procedure:

Male/Female ratio per cage: 1:1
Duration of cohabitation: 21 days
Proof of mating: vaginal plug or sperm in vaginal smear referred to as day 0 of presumed gestation.
If pairing was not successful after 14 days, replacement of first male by another male with proven fertility from the same dosage group. After successful conception, each pregnant female was individually housed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification of doses was carried with a Cary 118 twin beam spectrophotometer set at 211 nm.

Duration of treatment / exposure:

For F0 females: 96 - 130 daily doses
For F0 males: 93 - 94 daily doses
For F1 females: 104 - 149 daily doses
For F1 males: 106 - 125 daily doses

Frequency of treatment:

daily.

Details on study schedule:

All rats (male and female) were dosed with the test item Acrolein at 0, 1, 3 and 6 mg/kg at a volume of 5 ml/kg for 70 days. After the dosing period, rats within the same dosage group were housed together at a ratio of 1 male rat to 1 female rat for a minimum period of 14 days. Female rats that had not mated within the 14-day period were housed with a different male rat from the same dosage group for an additional 7 days. Copulatory plugs or spermatozoa in vaginal smears are observation was regarded as day 0 of the presumed gestation period. All female rats received their respective daily dose of Acrolein throughout cohabitation, gestation and lactation; this continued for all female rats until either day 25 of presumed gestation (for females who failed to deliver offspring) or day 21 of lactation (for females that successfully delivered offspring). F0 males were given daily doses up until the scheduled termination date.

F1 pups (40/sex from as many litters as possible/group, at least 1M + 1F/litter where possible) were selected for continuation of the study on day 21 of lactation. These pups received doses beginning on day 21 postpartum. F1 Female rats that had not mated within the 14-day period were housed with a different male rat from the same dosage group for an additional 7 days. When copulatory plugs or spermatozoa in vaginal smears are observed, this was regarded as day 0 of the presumed gestation period. All female rats received their respective daily dose of Acrolein throughout cohabitation, gestation and lactation; this continued for all female rats until either day 25 of presumed gestation (for females delivering no offspring) or day 21 of lactation (for females which successfully delivered offspring). F1 males were given daily doses up until the scheduled termination date.

F2 pups were not directly dosed with Acrolein (but may have been indirectly exposed).

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

30

Control animals:

yes

Details on study design:

A range-finding study was carried out with 6 groups of 16 rats (8 male, 8 female). All rats were dosed 7 days prior to cohabitation up to day 25 of presumed gestation or day 4 post partum. At the higher doses (10, 15 and 20 mg/kg), widespread mortality were observed. At doses of 5 mg/kg, clinical signs (including excess salivation, reduced motor activity and respiratory difficulty) were observed. No effect on mating or fertility parameters was seen at 2.5 mg/kg/day. Based on the results of the range-finding study, doses of 0, 1, 3, and 6 mg/kg were used in the main study.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day during study period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: daily during dosage period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- For males, this was recorded weekly (except during cohabitation - was recorded but not tabulated) For females, food consumption was recorded weekly prior to cohabitation on days 0, 6, 12, 15, 20 and/or 25 of presumed gestation; in addition on days 1, 4, 7, 10, 14, 16, 18 and 21 of lactation.

OTHER:
For all F0 and F1 female rats:
- The nursing behaviour, caring of pups and other dam-pup interactions were observed on a daily basis, any abnormal maternal behaviour was recorded.
- mating performance was assessed daily during the cohabitation period, confirmed by observed delivery of offspring, confirmed mating date or implantation site at necropsy.
- The duration of gestation and pup viability was assessed.
- Those that did not deliver offspring were killed on day 25 of presumed gestation and examined for implantation sites and gross lesions.

Necropsies were conducted on any deceased F0 and F1 rats.

Oestrous cyclicity (parental animals):

The oestrous cycle was assessed during the cohabitation period (maximum: 21 days) up until the presumed begin of gestation.

Litter observations:

STANDARDIZATION OF LITTERS
-Performed on day 4 postpartum: yes
-If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were removed
PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals: All surviving maternal animals on day 21 of lactation (for females that successfully delivered offspring) or day 25 of presumed gestation period (for females without offspring).

GROSS NECROPSY AND HISTOPATHOLOGY
- Gross necropsy and histopathology was performed on the following organs:
testes, seminal vesicles, epididymides, prostate, ovaries uterus, vagina, kidneys, lungs heart, liver, spleen, stomach, thyroid, pituitary glands, adrenal glands, spinal cord and brain.

Postmortem examinations (offspring):

SACRIFICE
The F1 offspring not selected as parental animals and all F2 offspring were sacrificed on day 21 post partum

GROSS NECROPSY AND HISTOPATHOLOGY
- Gross necropsy and histopathology was carried out on testes, seminal vesicles, epididymidis, prostate, ovaries uterus, vagina, kidneys, lungs heart, liver, spleen, stomach, thyroid, pituitary glands, adrenal glands, spinal cord and brain.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

details see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

details see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

details see below

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

details see below

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 6 mg/kg/day, P and F1 mortality was increased in both sexes (statistically significant except for P males). In both P and F1 parental generations, at the high dose level, the following significant clinical effects were observed: rales, gasping, hyperpnoea and irregular breathing. Other clinical effects observed (although not always significant) include, chromorhinorhea, yellow/red-brown perioral substance, bradypnoea, alopecia, excess salivation, abdominal distension, red/brown urine, soft/liquid stools or none at all, paleness, cold to the touch, emaciated appearance and head tilt.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In P and F1 generation males in the highest dose group, a significant decrease in weight gain was observed throughout the study . Highest dose F0 females also showed a decrease in body weight gain during the premating, gestation and lactation periods. High-dose F1 females also showed some
significant decreases in premating body weight.

Food consumption in the P and F1 generation males was reduced in the first 3 weeks of the study, but then increased throughout the remainder of the study (increased significantly compared to the control group). A similar effect was also seen with F0 and F1 females in the premating period.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No effect

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects on P and F1 generation male rats. However in F0 females, the reduced F1 pup body weights (high dose group) during the lactation period could be regarded as a reproductive effect but in conclusion this was not considered as a selective reproductive effect of acrolein.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No effects

GROSS PATHOLOGY (PARENTAL ANIMALS)
In P and F1 high-dose males, F1 females and F1 mid-dose females glandular and forestomach lesions were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In P and F1 high-dose males, F1 females and F1 mid-dose females lesions observed were hyperplasia/hyperkeratosis of the forestomach mucosa, increases of erosion(s), ulcer(s) and haemorrhage of the glandular mucosa and focal hyperplasia of the glandular mucosa. Increases in submucosal inflammation and edema with mononuclear-cell infiltrates were also seen in areas of erosion and ulcers. No effects were seen in reproductive organs

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Erosions of glandular mucosa and hyperplasia/hyperkreatosis of the forestomach

Dose descriptor:

NOAEL

Effect level:

> 6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no reproductive effects were observed

Remarks on result:

other: Generation: P and F1 (migrated information)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Reduction in body weights for F1 during lactation period at 6 mg/kg, but not considered as a selective reproductive effect of acrolein

Remarks on result:

other: Generation: P and F1 (migrated information)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: paternal (F0 and F1) based on stomach lesion and decreased body wheight gains

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
No effects
BODY WEIGHT (OFFSPRING)
F1 pup weights were significantly reduced in the high-dose group.
GROSS PATHOLOGY (OFFSPRING)
No effects
HISTOPATHOLOGY (OFFSPRING)
No effects

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: paternal (F0 and F1) based on stomach lesion and decreased body wheight gains

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced bodyweights during lactation at 6 mg/kg/day, not considered as a selective reproductive effect of acrolein

Key result

Critical effects observed:

no

Reproductive effects observed:

not specified

Allyl methacrylate will be hydrolyzed into allyl alcohol and methacrylic acid. Acrolein is a major toxic metabolite of allyl alcohol and therewith of allyl methacrylate.

Molecular weights:

Allyl methacrylate: 126.16 g/mole

Acrolein: 56.06 g/mole

Conclusions:

No study on 2-generation reproduction toxicity is available with Allyl methacrylate. Allyl methacrylate will be metabolized in a first step into allyl alcohol and methacrylic acid. Acrolein is knwn to be the toxic metabolite of allyl alcohol. Therefore the 2 -generation toxicity study in rats with arcolein will be used for the read across approach of the allyl alcohol moiety of allyl methacrylate.


Under the conditions of this study, the overall reproductive NOAEL is 6 mg/kg/d.

Executive summary:

No study on 2-generation reproduction toxicity is available with Allyl methacrylate. Allyl methacrylate will be metabolized in a first step into allyl alcohol and methacrylic acid. Acrolein is knwn to be the toxic metabolite of allyl alcohol. Therefore the 2 -generation toxicity study in rats with arcolein will be used for the read across approach of the allyl alcohol moiety of allyl methacrylate.

In a 2 -generation reproduction study in rats, acrolein was administered to 30 animals (Crl:CD (SD)BR strain) per sex and dose group by oral gavage at dose levels of 0, 1, 3 or 6 mg/kg for 70 days in a dosing volume of 5 ml/kg.

In both P and F1 parental generations, at the high dose level, mortality was increased and various clinical signs were observed (hyperpnoea, irregular breathing and other, minor signs). No impairment of reproductive performance on P and F1 generation male rats were observed.

However for F0 females (of the high dose group) reduced F1 pup body weights during the lactation period was not considered as a selective reproductive effect of acrolein.

No significant changes in reproductive organs were seen.

Histopathology findings in P and F1 high-dose males, F1 females and F1 mid-dose females were hyperplasia/hyperkeratosis of the forestomach mucosa, increases of erosions, ulcers and redness of the glandular mucosa and focal hyperplasia of the glandular mucosa. Increases in submucosal inflammation and edema with mononuclear-cell infiltrates were also seen in areas of erosion and ulcers.  Parental NOAEL was 1 mg/kg/day (based on erosions of glandular mucosa and hyperplasia/hyperkreatosis of the forestomach).

Male parental and F1 reproductive NOAEL was > 6 mg/kg/day, female parental and F1 reproductive NOAEL was 6 mg/kg/day and also male and female offspring NOAEL is 6 mg/kg/day as slight reduced body weight gains during lactation are not considered as reproductive effects in this study.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline studies.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Concerning reproduction there is a combined repeated-dose toxicity study with reproduction/developmental screening available (conducted in compliance with GLP, according to OECD 422).

In this study Allyl methacrylate was administered to Sprague-Dawley rats (10/sex/group) via gavage at 0 (corn oil), 3, 15, or 60 mg/kg bw/day. Males were treated once daily during pre-mating and the mating period for a minimum of 4 weeks. Females were treated once daily during pre-mating, mating and gestation and through post natal day (PND) 5 (the day of pup birth was designated PND 1). Males were sacrificed after the mating period, and females were sacrificed with their litters on post natal day (PND) 6. There were no treatment-related effects on mating, fertility indices, the duration of gestation, mean number of live born pups or on pup death after birth. There were no gross external pup abnormalities in the control or Allyl methacrylate-treated groups. The NOAEL for reproductive performance (mating and fertility) was 60 mg/kg bw/day (the highest dose tested).

As a general principle, the first step in Methacrylic acid ester metabolism is cleavage to methacrylic acid and the regarding alcohol. Consequently, toxicity of Allyl methacrylate can be approximately described by the toxicity of the cleavage products Allyl alcohol (or its relevant metabolite Acrolein) and Methacrylic acid and / or structural analogue Methacrylate esters.

Therefore, data were adopted by read-across from two-generation reproduction toxicity studies on methyl methacrylate and Acrolein. The latter is a relevant metabolite of Allyl alcohol and Allyl methacrylate, respectively.

The study on Methyl methacrylate was performed in rats with oral administration (gavage) according to OECD 416 with GLP. The NOAEL for general, systemic toxicity was determined to be 50 mg/kg bw/day for the P and F1 parental rats, based on adverse effects on food consumption observed at the LOAEL of 150 mg/kg bw/day in the P parental females. The NOAEL for fertility and reproductive performance for the P and F1 parental rats was determined to be 400 mg/kg bw/day, the highest dose tested. The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test Methyl methacrylate was determined to be 400 mg/kg bw/day, the highest dose tested.

Acrolein was administered to 30 animals (Crl:CD (SD)BR strain) per sex and dose group by oral gavage at dose levels of 0, 1, 3 or 6 mg/kg in a 2 -generation reproduction study in rats.

Parental NOAEL was 1 mg/kg/day (based on stomach lesions and reduction in body weight gain). Male parental and F1 reproductive NOAEL was > 6 mg/kg/day, female parental and F1 reproductive NOAEL was 3 mg/kg/day based on reduced body weight in parental offspring during lactation. Male and female offspring NOAEL is 3 mg/kg/day based on reduced weights.

Taken together, there are no indications that Allyl Methacrylate or analogous substances might have specific effects on reproduction/fertility to rats.

As no significant reproductive effects were observed in an OECD 416 2-generation reproduction oral toxicity study in rats with the main toxic metabolite of ally methacrylate were observed at 6 mg acrolein/kg/d, converted to allyl methacrylate a NOAEL of 13.5 mg allyl methacrylate/kg bw/d was calculated.

Effects on developmental toxicity

Description of key information

-Prenatal Developmental Toxicity Study (rat, inhalative): NOAEL (developmental toxicity): 50 ppm (0.262 mg/L/day) based on decreased fetal body weight at 100 ppm, NOAEL (maternal toxicity): not achieved (< 12 ppm [0.063 mg/L]) (similar to OECD 414)
-Reproduction/Developmental Toxicity Screening Test OECD 422 (rat, oral, gavage): NOAEL (maternal toxicity): 15 mg/kg bw/day, NOAEL (developmental toxicity): 60 mg/kg bw/day (according to OECD 422)

-

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-10-19 to 2004-12-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guidceline study, GLP

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test), adopted on 22 March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA guideline OPPTS 870.3650, July 2000

Deviations:

no

Principles of method if other than guideline:

The study was conducted as a combined repeated dose toxicity study with Reproduction / Developmental Toxicity Screening Test. In this part all parameters concerning Reproduction / Developmental Toxicity were considered.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: males were 8 weeks old and the females were 10 weeks old
- Weight at study initiation: 303 g (range: 280 g to 332 g) for the males and 229 g (range: 199 g to 273 g) for the females
- Fasting period before study: no
- Housing: The animals were housed individually (except during mating) in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust was placed under each cage. Shortly before parturition, the females were moved to polycarbonate cages (43.0 x 21.5 x 20.0 cm) with autoclaved sawdust and wood shavings as nesting material (SICSA, Alfortville, France).
- Diet: The animals had free access to SSNIFF R/M-H pelleted maintenance diet, Batch Nos. 2764132 and 4764439 (SNIFF Spezialdiäten GmbH, Soest, Germany) distributed weekly.
- Water: The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2004-10-12 To: 2004-12-07

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Vehicle
The vehicle was corn oil, Batch No. 103K0107, supplied by Sigma (Saint-Quentin-Fallavier, France).

Dosage formulation preparation
The test item dosage formulations were prepared by suspending Allyl Methacrylate (AMA) in corn oil in order to achieve the concentrations of 0.6, 3 and 12 mg/mL and were stored at +4°C or up to 9 days protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of the dosage formulations
Homogeneity
Two dosage formulations were prepared at 0.2 and 15 mg/mL of AMA (in corn oil) in order to cover all the concentrations intended for use in this study. From both dosage formulations, duplicate samples were taken at three different levels of the container (top, middle, bottom) and analyzed for concentration of the test item just after preparation (day 0) and after 9 days of storage at +4°C (protected from light).
Stability
The two dosage formulations prepared at 0.2 and 15 mg/mL of AMA in corn oil for homogeneity analysis were sampled after 0 (just after preparation), 4 and 9 days storage at +4°C (protected from light). The aliquots (n = 2) sampled on day 4 were stored frozen at -20°C pending analysis on the last sampling occasion, when all samples were assayed. The mean concentrations measured on days 0 and 9 for homogeneity were taken as the initial and final values for the stability test.
Concentration
The concentration of samples taken from each dosage formulation (including the control) prepared for use in weeks 1, 2, 4 and 7 was determined.

Results
A satisfactory agreement was observed between the nominal and actual concentrations of the test item in the dosage formulations analyzed since the deviations from nominal concentration remained in an acceptable range of ± 10%. Furthermore, there was a satisfactory correspondence between the nominal and the measured concentrations of the test item in the vehicle.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: during the 2 week mating period, one male and one female from the same dose group were co-habitated during the night until pregnancy occurs or the mating period is over
- Proof of pregnancy: vaginal plug / sperm in vaginal smear

Duration of treatment / exposure:

Females: 15 days before mating, through mating period, pregnancy and lactation, until day 5 post-partum inclusive (or until sacrifice, for unmated females);
Males: 15 days before mating, through mating and post-mating periods through sacrifice (minimum 4 weeks);

Frequency of treatment:

once daily

Duration of test:

Males: 15 days before mating, through mating and post-mating periods through sacrifice (minimum 4 weeks);
Females: 15 days before mating, through mating period, pregnancy and lactation, until day 5 post-partum inclusive (or until sacrifice, for unmated females);

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected following the results of a previously conducted study CIT/Study No. 28198 TSR in which rats were dosed (gavage) withAllyl Methacrylate (AMA), Batch. 00216X, at 0, 25, 75 and 150 mg/kg/day for 10 consecutive days. In this study, 150 mg/kg/day resulted in morbidity and mortality, both sexes in the 25 and 75 mg/kg/day groups showed signs of hypersalivation during the dosing period, males showed signs of piloerection at 75 mg/kg/day, and the females in the 75 mg/kg/day group had increased liver weight.
Taking into account that the duration of the dosing period for this study was nearly 6 weeks in females, the high dose-level was set at 60 mg/kg/day. The intermediate and low dose-levels were selected in order to identify a No Effect Level.

Maternal examinations:

- CLINICAL OBSERVATIONS: Clinical observations were conducted daily throughout the study period.

- DETAILE CLINICAL OBSERVATIONS (DCO): Conducted on all rats pre-exposure and weekly throughout the study. The DCOs included cage-side, handheld and open-field observations that were recorded categorically or using explicitly defined scales.

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded weekly throughout the study. Female body weights were performed weekly for the premating and mating periods. Maternal body weights were recorded on gd 0, 7, 14, 17 and 20. Females that delivered were weighed on ld 1 and 5. Females that failed to mate or deliver were weighed on a weekly basis for the study duration.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined weekly for males and females during the pre-mating period. Due to co-housing, food consumption was not measured during mating. During gestation, food consumption for the females was measured on gd 0, 7, 10, 14, 17 and 20. After parturition, food consumption was measured on ld 1 and 5.

OTHER:
Females were observed for signs of parturition beginning on gd 20. The first day the presence of a litter was noted was designated ld 1.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
- Number of implantations: Yes

Fetal examinations:

All litters were examined as soon as possible after delivery and the following information was recorded: litter size, the number of live and dead pups and the sex. Pup clinical observations and body weights were recorded on pnd 1 and 5. In addition, any physical abnormalities or demeanor changes were recorded during the lactation period.
Pups found dead and sacrificed on ld 6 were carefully examined for external gross abnormalities and a macroscopic evaluation was performed. All litters were examined as soon as possible after delivery.

Statistics:

The following sequence was used for the statistical analyses of body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data. If there were 3 or less animals/sex/group remaining, no statistical analysis was performed. If there were 5 or less animals/sex/group remaining for 3 or more groups, the data were analyzed using the Dunn test. If there was 5 or less animals/sex/group remaining for less than 3 groups, the data were analyzed using the Mann-Whitney/Wilcoxon test. If there was 5 or more animals/sex/group, the data were analyzed by Kolmogorov-Lillefors test to test for the normality of the distribution of the data. If it was not normal, the values were logarithmically transformed and retested for normality. If it still did not meet the criteria for normality, the original values were analyzed by the Dunn test. If the data were normal, and there were 3 or more groups, the data were assessed for homogeneity of variance between groups with the Bartlett test. If not homogeneous, the data were analyzed by the Dunn test; if homogeneous, the data were assessed by the Dunnett test. If the data were normal, and there was less than 3 groups, the data were assessed for homogeneity of variance between groups with the Fisher test. If not homogeneous, the data were assessed using the Mann-Whitney/Wilcoxon test; if homogeneous, the data were analyzed by the Student test. The statistical comparison of results of motor activity (horizontal andrearing movements recorded from five males and five females per group) was performed, according to the following steps: step 1: normality and homogeneity of variances (Kolmogorov-Smirnov and Bartlett test respectively), step 2: parametric (ANOVA plus Dunnett test if necessary) or non-parametric (Kruskal-Wallis plus Dunn test if necessary) tests was conducted based on the results obtained at step 1. These statistics were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc).

Indices:

The following calculations were made:
- Pre-implantation loss
- Post-implantation loss
- Mating index
- Fertility index
- Gestation index
- Live birth index
- Viability index

Historical control data:

no data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hypersalivation was observed in a dose-related manner in males and females given 15 or 60 mg/kg/day during the premating period (from week 2) and then in females during gestation and lactation. This clinical sign was not considered to be adverse as it likely represents a reaction to the dosing procedure and not a direct effect of AMA toxicity. At 60 mg/kg/day, one female experienced hypotonia and half-closed eyes on day 5 p.p.. There was no disturbance of autonomal or physiological functions at any dose-levels and the motor activity of the animals was considered to be unaffected by treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on group mean male body weight gain during the premating phase and only a marginal reduction in body weight gain was observed at 15 and 60 mg/kg/day (p<0.05) during the mating period

Haematological findings:

no effects observed

Description (incidence and severity):

There were no changes in hematological parameters that could be considered toxicologically relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For blood biochemistry investigation, one female dosed with 60 mg/kg/day had increased total bilirubin concentration and another one had increased biliary acid concentration, these changes were correlated with histopathological findings in the liver

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no effects of treatment on pH, specific gravity or volume of urine produced by the males in any group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The necropsy of the adult males and females revealed the presence of yellowish areas in the liver of 2/5 females in the 60 mg/kg/day dose group.
At histopathology, several foci of degenerated/necrotic hepatocytes, together with slight periportal fibrosis, slight biliary proliferation, and greenish-pigment-laden macrophages, were noted in 3/5 females given 60 mg/kg/day. There were no other treatment-related histopathological changes for either sex at any dose.

Dead fetuses:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the mean number of liveborn pups or on pup death after birth.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The duration of gestation was similar between the control and test item-treated groups and close to the normal value of 21 days

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There was no effect of treatment on mating at any dose-level.
The male and female fertility indices were unaffected by treatment; all mated females were pregnant with live fetuses

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on body weights of females during the premating period, gestation or lactation.
There were no external abnormalities in the control or test item-treated groups.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Hypersalivation was observed in a dose-related manner in males and females given 15 or 60 mg/kg/day during the premating period (from week 2) and then in females during gestation and lactation. This clinical sign was not considered to be adverse as it likely represents a reaction to the dosing procedure and not a direct effect of AMA toxicity. At 60 mg/kg/day, one female experienced hypotonia and half-closed eyes on day 5 p.p.. There was no disturbance of autonomal or physiological functions at any dose-levels and the motor activity of the animals was considered to be unaffected by treatment. There was no effect of treatment on group mean male body weight gain during the premating phase and only a marginal reduction in body weight gain was observed at 15 and 60 mg/kg/day (p<0.05) during the mating period. There was no effect of treatment on body weights of females during the premating period, gestation or lactation. There was no effect of treatment on food consumption for females during the premating period, gestation or lactation. There were no changes in hematological parameters that could be considered toxicologically relevant.
For blood biochemistry investigation, one female dosed with 60 mg/kg/day had increased total bilirubin concentration and another one had increased biliary acid concentration, these changes were correlated with histopathological findings in the liver. There were no effects of treatment on pH, specific gravity or volume of urine produced by the males in any group.
-Mating Data: There was no effect of treatment on mating at any dose-level.
-Fertility Data: The male and female fertility indices were unaffected by treatment; all mated females were pregnant with live fetuses.
-Delivery Data: The duration of gestation was similar between the control and test item-treated groups and close to the normal value of 21 days. There was no effect of treatment on the mean number of liveborn pups or on pup death after birth. There were no external abnormalities in the control or test item-treated groups.
The necropsy of the adult males and females revealed the presence of yellowish areas in the liver of 2/5 females in the 60 mg/kg/day dose group.
At histopathology, several foci of degenerated/necrotic hepatocytes, together with slight periportal fibrosis, slight biliary proliferation, and greenish-pigment-laden macrophages, were noted in 3/5 females given 60 mg/kg/day. There were no other treatment-related histopathological changes for either sex at any dose.

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no effect of treatment on the mean number of liveborn pups or on pup death after birth.
There were no external abnormalities in the control or test item-treated groups.

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Remarks on result:

other: No relevant findings were observed in the pups sacrificed on day 6 postpartum

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In this study, the NOAEL for developmental toxicity was 60 mg/kg bw/day (the highest dose tested).

Executive summary:

In this combined repeated-dose toxicity study on Allyl methacrylate with reproduction/developmental screening according to OECD TG 422, there were no effects on mean number of live born pups or pup death after birth. Male and female pups at 15 or 60 mg/kg bw/day gained less weight than the controls but the changes did not reach statistical significance. The number of pups that were cold to the touch was higher at 60 mg/kg bw/day than in the control litters. No relevant findings were observed in the pups sacrificed on day 6 postpartum. Based on liver effects the NOAEL for maternal toxicity was 15 mg/kg bw/day. The NOAEL for developmental toxicity was 60 mg/kg bw/day (the highest dose tested).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l'Arbresle, France)
- Weight at study initiation: 180-200 g at study initiation
- Housing: single (cohousing only throuh mating period)
- Diet: Food pellets ad libitum except during exposures (UAR Alimentation ViIlemoisson, France)
- Water: filtered tap water ad libitum(except during exposures)
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow
- System of generating particulates/aerosols: The vapor generation system consisted in passing an additional airflow rate through the fritted disk of a heated bubbler containing the test chemical. The vaporized compound was introduced into the main air-inlet pipe of the exposure chamber.
- Temperature, humidity, pressure in air chamber: In order to prevent any leakage of the test atmospheres the chambers were maintained at a negative pressure of no more than 3-mm water. The chamber temperature was set at 23+/-2°C and the relative humidity at 50 ± 5% .
- Air flow rate: 6-20 m3/h

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatograph

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber atmospheres were monitored continually with a gas chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. Additionally, the exposure levels were determined once during each 6-hour exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal and analyzed by gas chromatography. The charcoal samples were then desorbed with carbon disulfide. The resulting samples were then analyzed by gas chromatography using appropriate internal standards.

Details on mating procedure:

- M/F ratio per cage: 1 male : 2 or 3 females from the same strain and supplier
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from gestation days (GD) 6-20

Frequency of treatment:

6 h per day

Duration of test:

from mating to caesarean section (GD 21)

No. of animals per sex per dose:

23-27 bred rats/group (19-25 pregnant/group)

Control animals:

yes, concurrent no treatment

Details on study design:

no data

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: GDs 0, 6, 13, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes (for the intervals GDs 6-13 and 13-21)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: uterus

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
- Other: dead and live fetuses

Fetal examinations:

- External examinations: Yes (all per litter)
- Soft tissue examinations: Yes (half per litter)
- Skeletal examinations: Yes (half per litter)
Live fetuses were weighed and sexed.

Statistics:

Whenever possible, the data were presented as mean ± SD. The number of corpora lutea, implantation sites and live fetuses, maternal food consumption, and various body weights were analyzed by one-way analysis of variance, followed by Dunnett's test if differences were found. The percentages of non-live implants, resorptions, and males, and the proportions of fetuses with alterations in each litter were evaluated by the Kruskal-Wallis test, followed by the Dixon-Massey test where appropriate. Rates of pregnancy and percentages of litters with any malformations or external, visceral, or skeletal variations were analyzed by using Fisher's test. Where applicable, least-squares analysis was carried out. The reported level of statistical significance was p< 0.05. The litter was used as the basis for analysis of fetal variables.

Indices:

The percentages of non-live implants, resorptions, and males, and the proportions of fetuses with alterations in each litter were calculated.

Historical control data:

no data

Mortality:

no mortality observed

Description (incidence):

All female rats survived the test period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal weight gain was significantly less than control during the first half of exposure at 12 and 25 ppm and throughout the entire exposure period at 50 and 100 ppm. The overall weight gain on GD 6-21 was significantly reduced at 12 ppm and higher concentrations. When GD 21 body weights were corrected for gravid uterine weight, weight gain over the exposure period was severely reduced at 12 and 25 ppm, and body weight loss occurred at 50 and 100 ppm.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

No data on food consumption was available because of a technical failure

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

The mean number of implantation sites and of live fetuses was comparable across groups. There was a slight and non-significant increase in the incidence of non-live implants and of resorptions at 50 and 100 ppm, compared to the concurrent control.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: reduced body weight and body weight gain

Details on maternal toxic effects:
All female rats survived the test period.
Maternal weight gain was significantly less than control during the first half of exposure at 12 and 25 ppm and throughout the entire exposure period at 50 and 100 ppm. The overall weight gain on GD 6-21 was significantly reduced at 12 ppm and higher concentrations. When GD 21 body weights were corrected for gravid uterine weight, weight gain over the exposure period was severely reduced at 12 and 25 ppm, and body weight loss occurred at 50 and 100 ppm.
No data on food consumption was available because of a technical failure.
The mean number of implantation sites and of live fetuses was comparable across groups. There was a slight and non-significant increase in the incidence of non-live implants and of resorptions at 50 and 100 ppm, compared to the concurrent control.

Key result

Dose descriptor:

NOAEL

Effect level:

< 0.063 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

LOAEL

Effect level:

0.063 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

0.263 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEL

Effect level:

0.524 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a concentration-related decrease in fetal body weight, which achieved statistical significance at 100 ppm (10% lower than control).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations were observed in one control fetus and in two fetuses from the low-concentration group. Several common visceral and skeletal variations were observed with no statistically significant differences between treated and control groups.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: concentration-related decrease in fetal body weight

Details on embryotoxic / teratogenic effects:
There was a concentration-related decrease in fetal body weight, which achieved statistical significance at 100 ppm (10% lower than control).
Visceral malformations were observed in one control fetus and in two fetuses from the low-concentration group. Several common visceral and skeletal variations were observed with no statistically significant differences between treated and control groups.
No compound-induced teratogenic effects were observed.

Dose descriptor:

NOAEC

Effect level:

0.262 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

other: decreased fetal body weight at 0.524 mg/l

Abnormalities:

no effects observed

Developmental effects observed:

no

Weight change during gestation
Concentration ppm/6 h/day	No. of dams (a)	Body weight (g) on GD 6	Body weight gain (g) on GD			Absolute weight gain (g)
			6-13	13-21	6-21
0	20	268+/-16	35+/-6	95+/-17	130+/-20	29+/-11
12.5	19	271+/-23	19+/-8**	89+/-20	108+/-22*	3+/-11**
25	21	269+/-11	17+/-10**	80+/-15	98+/-18**	2+/-9**
50	20	272+/-20	9+/-7**	72+/-26**	81+/-28**	-5+/-15**
100	21	271+/-17	5+/-7**	65+/-29**	70+/-33**	-11+/-15**
(a) includes all dams pregnant at euthanization
*,** significant differences from the control (0 ppm) value, p < 0.05 and p < 0.01, respectively

Reproductive parameters on days 6 to 20 and euthanized on day 21

Concentration ppm/6 h/day

Test animals (dams)

Litters with implants

Litters with live fetuses

No. of litters

No. of live fetuses per litter

% of males per litter

Average fetal body weight (g) per litter

No. of dead/no of treated

% of females pregnant at euthanization

No. of corpora lutea per dam

No. of implantation sites per litter

% of nonlive implants per litter (a)

% of resorption sites per litter

All

Males

Females

0

0/23

87

20

15.55 +/- 2.28

14.70 +/- 2.89

7.39 +/- 6.56

20

13.60+/- 2.87

54.81 +/- 12.61

5.55 +/- 0.26

5.69 +/- 0.25

5.37 +/- 0.31

12.5

0/23

82.6

19

16.05 +/- 2.04

15.26 +/- 2.26

5.91 +/- 8.85

19

14.42 +/- 2.67

48.31 +/- 14.88

5.44 +/- 0.23

5.57 +/- 0.29

5.31 +/- 0.22

25

0/23

91.3

21

15.38 +/- 1.63

14.76 +/- 1.92

8.32 +/- 9.56

21

13.67 +/- 2.63

49.94 +/- 11.03

5.44 +/- 0.34

5.51 +/- 0.39

5.31 +/- 0.38

50

0/23

87

20

15.79 +/- 2.02

14.15 +/- 4.38

12.15 +/- 23.63

19

13.37 +/- 4.50

52.37 +/- 20.84

5.33 +/- 0.64

5.46 +/- 0.62

5.11 +/- 0.52

100

0/23

91.3

21

15.68 +/- 1.86

14.38 +/- 3.65

18.63 +/- 29.16

19

13.42 +/- 3.20

54.75 +/- 14.62

5.01 +/- 0.25**

5.14 +/- 0.27**

4.82 +/- 0.34**

(a) Resorptions plus dead fetuses.

*,** denote significant differences from the control (0 ppm) value, p < 0.05 and p < 0.01, respectively.

Conclusions:

In this study, the NOAEL for developmental toxicity was 50 ppm and for teratogenicity 100 ppm, the highest dose tested.

Executive summary:

An inhalative prenatal developmental toxicity study was conducted with Allyl methacrylate similar to OECD 414. Pregnant Sprague-Dawley rats (19-25 / concentration) were exposed to Allyl methacrylate vapour via whole body inhalation for 6 h per day during gestation days 6-20 at 0, 12, 25, 50, and 100 ppm (0, 0.063, 0.131, 0.262, 0.524 mg/L). Maternal weight gain was reduced at and above 12 ppm compared to the control. The mean number of implantation sites and number of live fetuses was comparable across groups. There was a concentration-related decrease in fetal body weight that achieved statistical significance at 100 ppm (10% lower than control). There was a slight and non significant increase in the incidence of non-live implants and of resorptions at 50 and 100 ppm, compared to the concurrent control. Several common visceral and skeletal variations were observed with no statistically significant differences between treated and control groups. No compound-induced teratogenic effects were observed. The NOAEC for maternal toxicity was not achieved (< 12 ppm [0.063 mg/L]) and the NOAEC for developmental toxicity was 50 ppm (0.262 mg/L/day) based on decreased fetal body weight at 100 ppm.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Quality of whole database:

GLP and guideline study.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

262 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Comparable to guideline study with acceptable restrictions.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Allyl methacrylate has been tested in reliable developmental toxicity studies in rats (inhalative and oral route).

Inhalative:

An inhalative prenatal developmental toxicity study was conducted with Allyl methacrylate similar to OECD 414. Pregnant Sprague-Dawley rats (19-25 / concentration) were exposed to Allyl methacrylate vapour via whole body inhalation for 6 h per day during gestation days 6-20 at 0, 12, 25, 50, and 100 ppm (0, 0.063, 0.131, 0.262, 0.524 mg/L). Maternal weight gain was reduced at and above 12 ppm compared to the control. The mean number of implantation sites and number of live fetuses was comparable across groups. There was a concentration-related decrease in fetal body weight that achieved statistical significance at 100 ppm (10% lower than control). There was a slight and nonsignificant increase in the incidence of non-live implants and of resorptions at 50 and 100 ppm, compared to the concurrent control. Several common visceral and skeletal variations were observed with no statistically significant differences between treated and control groups. No compound-induced teratogenic effects were observed. The NOAEC for maternal toxicity was not achieved (< 12 ppm [0.063 mg/L]) and the NOAEC for developmental toxicity was 50 ppm (0.262 mg/L/day) based on decreased fetal body weight at 100 ppm (Saillenfait, 1999).

Oral:

In the combined repeated-dose toxicity on Allyl methacrylate study with reproduction/developmental screening test (CIT, 2009), there were no effects on mean number of live born pups or pup death after birth. Male and female pups at 15 or 60 mg/kg bw/day gained less weight than the controls but the changes did not reach statistical significance. The number of pups that were cold to the touch was higher at 60 mg/kg bw/day than in the control litters. No relevant findings were observed in the pups sacrificed on day 6 postpartum. Based on liver effects the NOAEL for maternal toxicity was 15 mg/kg bw/day. The NOAEL for developmental toxicity was 60 mg/kg bw/day (the highest dose tested).

Taken together, there are no indications that Allyl Methacrylate might cause developmental / teratogenic effects even applied at systemic toxic doses in either rats or rabbits.

According Annex IX the requirement of a developmental toxicity study (OECD 414) at one species is fulfilled. As no developmenta/teratogenic effects have been observed a study on a second specie is not requierd at this tonnage band.

Justification for classification or non-classification

- Oral exposure of Allyl methacrylate in rats did not result in toxicity reproductive or developmental toxicity at doses as high as 60 mg/kg bw/day in an oral combined repeated-dose/reproductive/developmental toxicity screening study.

- No significant reproductive effects were observed in an OECD 416 2-generation reproduction oral toxicity study in rats with the main toxic metabolite of ally methacrylate were observed at 6 mg acrolein/kg/d.

Converted to allyl methacrylate a NOAEL of 13.5 mg allyl methacrylate/kg bw/d was calculated.

- In an OECD 414 study with inhalative exposure, no effects with regard to development of the pubs were noted up to 0.262 mg/L, causing clear systemic toxicity in the dams. At 0.524 mg/L some effects on fetal body weight were noted. Within this study, a NOAEC of 0.262 mg/L for developmental effects and a NOAEC of 0.063 mg/L for maternal toxicity were deduced. Moreover, studies on analogue substances and relevant metabolites showed no specific effects on fertility or developmental toxicity.

In conclusion, Allyl methacrylate has not to be classified according Regulation (EC) No 1272/2008 (CLP).