Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study. Read across was performed with methyl methacrylate.
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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
Remarks:
Doses / Concentrations:
0, 50, 150, 450 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
F0 generation parental animals: 25
F1 generation parental aniumals: 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.
Test group 03 (400 mg/kg bw/d):
P PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY
- 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%)

F1 PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- 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 17%)

Test group 02 (150 mg/kg bw/d):
P PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- 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%)

F1 PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d):
P PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- no test substance-related adverse effects/findings

F1 PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- no test substance-related adverse effects/findings
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P and F1 parental animals (migrated information)
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Generation: P and F1 parental animals (migrated information)
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Generation: F1 and F2 progeny (migrated information)
Test group 03 (400 mg/kg bw/d):
F1 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

F2 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

Test group 02 (150 mg/kg bw/d):
F1 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

F2 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d):
F1 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

F2 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings
Reproductive effects observed:
not specified

- 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

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Reliable and guideline compliant study conducted with a suitable read-across substance
Additional information

Studies of reproduction and developmental toxicity of t-BMA are not available. Reliable studies of analogous substances were used to assess the potential of t-BMA for reproduction toxicity.

Data availability:

Regarding fertility, there is screening-level information on nBMA (OECD 422 study). Sufficient data to address all reproductive endpoint can be obtained by read-across from a 2-generation reproductive toxicity study on MMA by the oral route. No further testing is proposed.

While there is no full reproduction study with n-BMA, Methyl methacrylate has recently been tested in an OECD TG 416 oral two-generation reproduction toxicity study in rats, in which both, parental and F1 animals were dosed with 0; 50; 150 and 400 mg/kg body weight/day (BASF, 2010).

In summary, in mid- and high-dose parental animals (150 and 400 mg/kg bw/d) temporary salivation, presumably due to a bad taste of the test substance and associated dose-related intermittent reductions of food consumption were noted. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group and not associated with effects on histopathology or reproductive performance. 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 substance was determined to be 400 mg/kg bw/day, the highest dose tested. There were no signs of systemic toxicity other than reduced body weight gain associated with reduced food consumption, presumably due to bad palatability (NOEL 50 mg/kg bw/day for the P and F1 parental rats and LOEL of 150 mg/kg bw/day in the P parental females).

In detail: 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 adversely 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 histopathological 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 histopathological 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 behaviour, 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-/foetal 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). 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 substance was determined to be 400 mg/kg bw/day, the highest dose tested.

In an OECD Guideline 422 screening study, n-BMA in sesame oil was given by oral gavage to 10 male and 10 female rats at doses of 0, 30, 100, 300, or 1000 mg/kg/day (Ito et al., 1998). Male rats were dosed for 44 days and female rats were dosed from 14 days prior to mating through Day 3 of lactation. Treatment-related decreases in body weight and food consumption were observed in high dose (1000 mg/kg/day) animals only. Relative to reproductive performance, there were no treatment-related effects on any endpoint of mating or fertility at any dose level. There were no treatment-related effects on gestation index, gestation length or number of pups per litter. There were no treatment-related deaths during delivery, nor treatment-related effects on offspring viability or the ratio of male to female pups. There were no adverse effects on the reproductive organs at any dose of male (testes, epididymides, prostate, seminal vesicles) and female (ovaries, uterus, cervix, vagina) rats shown by gross and histopathological examination. Observed decreases in numbers of corpora lutea and implantations in parental females in the high dose group were the only effects ascribed to treatment.

Thus, based on effects observed in parental females in the 1000 mg/kg/day dose group, the NOAEL for reproductive toxicity is considered to be 1000 mg/kg/day for parental males and 300 mg/kg/day for parental females.


Short description of key information:
In a combined repeated dose and reproductive/developmental toxicity screening test with the read-across substance n-butyl methacrylate the reproductive and developmental toxicological NOAEL is considered as 1000 mg/kg/day for parental males and offspring, and 300 mg/kg/day for parental females since decreases in the number of corpora lutea and implantation sites were observed in the 1000 mg/kg group.

In a two generation reproduction toxicity the NOAEL for general, systemic toxicity was determined as 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 substance was alos determined to be 400 mg/kg bw/day, the highest dose tested.

Justification for selection of Effect on fertility via oral route:
Higher tier study - 2 Generation study in rats

Effects on developmental toxicity

Description of key information
Inhalation
In a prenatal development toxicity study (inhalation route) conducted with the the read-across test item n-butyl methacrylate in rats the NOAEL for developmental toxicity was 300 ppm. There was no evidence of embryolethality or teratogenicity with n-BMA. 
In a prenatal development toxicity study (inhalation route) conducted with the read-across test item methyl methacrylate in rats, no embryo of fetal toxicity was evident and no increase in the incidence in the malformations or variations was noted. The NOAEC for teratogenicity and fetotoxicity were considered to be 8.3 mg/L and the LOEC maternal toxicity was considered to eb 0.41 mg/L.
Oral exposure
In a prenatal development toxicity study conducted with the read-across test item n-butyl methacrylate in rabbits the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/day. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg bw/day. There were no adverse fetal findings evident at a dose not producing maternal toxicity. The compound is not regarded as teratogen.
In a prenatal development toxicity study conducted with the read-across test item methyl methacrylate in rabbits the no observed adverse effect level (NOAEL) for maternal toxicity is 50 mg/kg bw/day. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 450 mg/kg bw/day. There were no adverse fetal findings evident at a dose not producing maternal toxicity. The compound is not regarded as teratogen.
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study. Read across was performed with butyl methacrylate.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: 18-23 weeks
- Weight at study initiation: 1901-2589 g
- Fasting period before study: no
- Housing: the rabbits were housed singly in type 12.2395.C stainless steel wire mesh cages supplied by Draht-Bremer GmbH, Marktheidenfeld, Germany (floor area about 3,000 cm²)
- Diet: pelleted “Kliba maintenance diet for rabbits & guinea pigs, GLP, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. to 6.00 a.m. dark, 6.00 a.m. to 6.00 p.m. light)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at daily intervals, which took into account the analytical results of the stability verification. For the test substance preparation, an specific amount of test substance was weighed depending on the dose group, into a graduated flask, topped up (shortly under the marking) with 1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The graduated flask was sealed and the preparation was intensely mixed with a magnetic stirrer. During administration, the
preparations were kept homogeneous with a magnetic stirrer.
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 twice during the study period (at the beginning and towards the end) for verification of the concentrations. Samples were analyzed by GC with external calibration.
Details on mating procedure:
The animals were paired by the breeder (time-mated animals), the day of pairing was designated as gestation day (GD) 0. Presumed pregnant animals were supplied one day after mating; this day was referred to as GD 1 and the following day as GD 2.
Duration of treatment / exposure:
from implantation to one day prior to the expected day of parturition (GD 6-28)
Frequency of treatment:
daily
Duration of test:
until GD 29
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
25 time-mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose volume was 10 mL/kg body weight. The calculation of the volume administered was based on the most recent individual body weight.
Maternal examinations:
CLINICAL EXAMINATIONS
- Mortality: Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 1-29).
- Clinical symptoms: A clinical examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 1-29).
- Food consumption: The food consumption was determined daily on GD 2–29.
- Body weight data: All animals were weighed on GD 1, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated.
- Corrected (net) body weight gain: The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

TERMINAL EXAMINATIONS OF THE DOES
After the does had been sacrificed on GD 29, they were necropsied and assessed by gross pathology in randomized order.
Ovaries and uterine content:
On GD 29, the surviving does were sacrificed in randomized order by an intravenous injection of pentobarbital (Narcoren; dose: 2 mL/animal). All prematurely dead or sacrificed females were examined following the same procedures as for females sacrificed on schedule with the exception that no uterine weights were determined.
After the does had been sacrificed, they were necropsied and assessed by gross pathology in randomized order. The uterus and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
1) live fetuses
2) dead implantations: a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy); b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible); c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).
After the weight of the uterus had been determined, all subsequent evaluations of the does and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose the animal numbers were encoded.
Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out:
The conception rate (in %) was calculated according to the following formula: (number of pregnant animals)/(number of fertilized animals) x 100.
The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula: (number of corpora lutea – number of implantations)/(number of corpora lutea) x 100.
The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula: (number of implantations – number of live fetuses)/(number of implantations) x 100.
Fetal examinations:
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
- Examination of the fetuses after dissection from the uterus: Each fetus was weighed and examined macroscopically for any external findings.
Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren; 0.2 mL/fetus).
- Soft tissue examination of the fetuses: After the fetuses had been sacrificed, the abdomen and the thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure. The sex of the fetuses was determined by examination of the gonads in situ. After these examinations, the heads of approximately one half of the fetuses per litter and the heads of those fetuses, which revealed severe findings during the external examination (e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN's solution and were, after fixation, processed and evaluated according to WILSON's method (Wilson and Warkany, 1965). About 10 transverse sections were prepared per head. After the examination these heads were discarded. All fetuses (partly without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the fetuses were removed from the fixative for awhile. With a scalpel, a transversal incision was made into the frontal / parietal bone in the heads of the intact fetuses. The two halves of the calvarium were then cauteously bent outward and the brain was thoroughly examined. Subsequently, the fetuses were placed back into the fixative for further fixation.
- Skeletal examination of the fetuses: After fixation in ethyl alcohol the skeletons were stained according to a modified method of KIMMEL and TRAMMELL (Kimmel, C.A., and Trammell, C., 1981). Thereafter, the stained skeleton of each fetus was examined. After the examination the stained skeletons were retained individually.
Statistics:
- DUNNETT-test (two-sided) for food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
- FISHER'S EXACT test (one-sided) for female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
- WILCOXON-test (one-sided) for proportions of fetuses with malformations, variations and/or unclassified observations in each litter.
Details on maternal toxic effects:
Details on maternal toxic effects:
- Mortality: Seven high-dose females had to be sacrificed after abortion on several days towards the end of the treatment period (GD 24-28). Before abortion, the food consumption in all affected individuals was distinctly reduced and they showed adverse clinical symptoms like reduced and no defecation. These 7 premature sacrifices because of abortions are considered to be a consequence of test substance-induced maternal toxicity. There were no test substance-related or spontaneous mortalities in any other group.
- Clinical symptoms: Test substance-associated clinical findings such as reduced/no defecation and abortions were observed in the high-dose group. Although such findings are not uncommon in rabbits, they were, in these particular cases, most likely a consequence of the massively reduced food consumption in these rabbits. Taking this fact and the high incidence of findings in the high-dose group into account they are thus considered to be related to the treatment. By contrast the low frequency of findings like reduced defecation or blood in bedding does not suggest a relationship to the treatment in the low- and mid-dose groups.
- Food consumption: The food consumption in the high-dose females (1000 mg/kg bw/d) was distinctly and statistically significantly reduced during the almost entire treatment period (GD 6-27 and 28-29). During the treatment period (GD 6-28) the total average food consumption of the highdose
rabbits was about 44% below controls. Additionally, a particularly massive decrease of individual food consumption was noted for all high-dose does which aborted. During the days prior to abortion the food consumption was almost disrupted in these animals. The females of the mid-dose group (300 mg/kg bw/d) also had a statistically significant lower food consumption in a number of intervals during the treatment (GD 8-9, 13-16, 17-18, 19-20, 21-22, and 26-29). During the treatment period (GD 6-28) the total average food consumption of the mid-dose rabbits was about 18% below controls. The food consumption of the low-dose does (100 mg/kg bw/d) did not show test substance-related impairments. The reduced food consumption at the 300 and 1000 mg/kg bw/d levels is considered to be related to the treatment.
- Body weight data: The mean body weights of the high-dose rabbits (1000 mg/kg bw/d) were statistically significantly decreased on GD 21, 23, 25 and 29. At termination (GD 29) the body weight of the surviving pregnant high-dose rabbits was about 5% below the concurrent control. The average body weight gain of these rabbits was also statistically significantly reduced at a number of treatment intervals (GD 6-9, 16-19, 19-21, 25-28). The mean body weight gain of the high-dose rabbits was significantly reduced by about 47% during the treatment period. The mean body weights and the mean body weight gain of the low- and mid-dose rabbits (100 and 300 mg/kg bw/d) were not significantly different from the concurrent controls throughout the study.
- Corrected (net) body weight gain: Mean carcass weights were comparable among all groups. The corrected (net) body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) was statistically significant (about 54 %) below the concurrent control at the high-dose (1000 mg/kg bw/d) level. Although not statistically significant the net body weight gain was still 37% below control at the mid-dose (300 mg/kg bw/d) level. These reductions in net body weight gain arer considered to be related to the treatment. No adverse effect on net body weight gain was noted in the low-dose group.
- Uterus weight: The mean gravid uterus weight in the high-dose group (1000 mg/kg bw/d) was statistically significantly reduced (about 21% below the control). The mean gravid uterus weights of test groups 1 and 2 (100 or 300 mg/kg bw/d) did not show statistically significant differences in comparison to the control group.
- Necropsy findings: At necropsy, 7/25 high-dose does had stomach erosions, 5/25 high-dose does had no feces in the small intestine and 3/25 high-dose does had watery feces in the intestines. These findings are related to the markedly reduced food consumption and are considered to be treatment-related. In the mid- and low-dose groups, only incidental, not test substance-related findings were noted in single females of each of these test groups.
- Reproduction data of does: The conception rate was 100% in all test groups including controls (0; 100; 300 or 1000 mg/kg bw/d). Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 18-25 pregnant rabbits per group had implantation sites in the uterus, at terminal sacrifice. The resorption rate (and hence the postimplantation loss) was statistically significant higher in the high-dose group (1000 mg/kg bw/d). This apparent effect was mainly caused by two does of this dose group which had only resorptions but no viable fetuses in the uterus and contributed to the average with 100% resorption rates. The litter losses in these two does are considered to be a direct consequence of maternal toxicity. In particular the markedly reduced food consumption after mid-gestation affected these animals in the same manner as the animals with abortions. All other does of the high-dose group did not show any changes of gestational parameters which were outside the normal range for this strain. There were no test substance-related and/or biologically relevant differences between the low- and mid-dose groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age; see also Part III (SUPPLEMENT) for historical control data.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
- Sex distribution of fetuses: The sex distribution of the fetuses in test groups 1-3 (100; 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.
- Weight of placentae: The mean placental weights in test groups 1, 2 and 3 (100; 300 and 1000 mg/kg bw/d) were comparable to the controls.
- Weight of fetuses: The mean fetal weight of the high-dose group (1000 mg/kg bw/d) was statistically significant below the corresponding control value (-10%). The mean fetal weights of the low- and mid-dose groups (100 or 300 mg/kg bw/d) were not influenced by the test substance. Neither female nor male weights showed statistically significant or biologically relevant differences between these groups and the controls.
- Fetal external malformations: External malformations, some associated with corresponding soft tissue and skeletal malformations, were recorded for single fetuses of all test groups including controls (0; 100; 300 and 1000 mg/kg bw/d). With the exception of cleft palate all external malformations were present in the historical control data. Each of the findings affected individual fetuses and neither statistically significant differences between the test groups nor a dose-response relationship were observed. No malformation pattern was evident. Thus an association of these individual findings to the treatment is not assumed. The total incidences of external malformations were comparable to the historical control data.
- Fetal external variations: One external variation (paw hyperflexion) occurred in single fetuses of the mid- and high-dose groups and the control. The incidences did not demonstrate a dose-response relationship and were comparable to the historical control data. Thus an association of this finding to the treatment is not assumed.
- Fetal external unclassified observations: No unclassified external observation was recorded in this study.
- Fetal soft tissue malformations: The examination of the soft tissues revealed a variety of malformations in fetuses of all test groups including the controls (0; 100; 300 and 1000 mg/kg bw/d). All individual soft tissue malformations were present in the historical control data at comparable frequencies. Neither statistically significant differences between the test groups nor a dose-response relationship were observed. No malformation pattern was evident. Thus an association of these findings to the treatment is not assumed.
- Fetal soft tissue variations: A number of soft tissue variations such as absent lung lobe (lobus inferior medialis) and malpositioned carotid branch was detected in each test group including the controls. Incidences were without a relation to dosing. Neither statistically significant differences between the test groups nor differences to the historical control data were noted.
- Fetal soft tissue unclassified observations: Unclassified soft tissue observations, such as infarct of liver, discolored kidney, hemorrhagic ovary and blood coagulum around urinary bladder, were recorded for some fetuses of test groups 0, 1 and 2 (0; 100 and 300 mg/kg bw/d). A relation to dosing is not present. Therefore, a test substance induced effect is not assumed.
- Fetal skeletal malformations: Malformations of the fetal skeletons were noted in all test groups including controls (0; 100; 300 and 1000 mg/kg bw/d). All individual skeletal malformations were present in the historical control data. With the exception of severely fused sternebrae (bony plate), which was observed at a slightly higher incidence, the frequencies of those malformations were comparable to the historical control data. With regard to the total malformation incidences, in particular the litter incidence, neither statistically significant differences between treated groups and control nor a dose-response relationship were observed.
- Fetal skeletal variations: For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were statistically significant higher exclusively in the
high dose group on a fetus per litter basis. Several specific skeletal variations were statistically significant higher than the concurrent and outside the historical control in the high dose group (on a fetus per litter basis). These findings are delays or minor disturbances of ossification which are reversible or do not considerably affect the integrity of the underlying structures. Such slight changes of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain, but considering the overall higher rate of skeletal variations in the top-dose group they may be correlated to the treatment.
- Fetal skeletal unclassified cartilage observations: Additionally, isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing and were considered to be spontaneous in nature.
- Abstract of all classified fetal external, soft tissue and skeletal observations: Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. The most salient of these malformations affected the head/skull (malformed skull bones, cleft palate), the heart/great vessels (various defects), gall bladder (absent), kidneys (absent, small) and the sternebrae (fused to a bone plate). All malformations are present in the historical control data, with the exception of cleft palate. No dose-response relationship is evident for the individual malformations. At the low- and mid-dose levels (100 and 300 mg/kg bw/d) the overall incidences are comparable to the concurrent and historical controls, whereas the overall incidence is slightly above the controls at the high-dose level. Importantly, as a number of morphological structures of different ontogenic origin is affected these various fetal findings do not form a distinct and consistent malformation pattern. One external (paw hyperflexion), two soft tissue (absent lobus inferior medialis and malpositioned carotid branch) and a broad range of skeletal variations occurred in all test groups including the controls. At the low- and mid-dose levels (100 and 300 mg/kg bw/d) all fetal and litter incidences for these variations and the corresponding rates of affected fetuses/litter were comparable to the control whereas at the high-dose level a slightly higher rate of affected fetuses per litter was noted. However, all incidences including the high-dose incidence can be found at a comparable frequency in the historical control data. A spontaneous origin is assumed for soft tissue and unclassified skeletal cartilage observations, which were observed in several fetuses of test groups 0, 1, 2 and 3 (0; 100; 300 and 1000 mg/kg bw/d) and the control. Distribution and type of these findings did not suggest relation to treatment.
Abnormalities:
not specified
Developmental effects observed:
not specified

Table: Postimplantation loss and resorption data

 

 

Group 0

0 mg/kg/d

Group 1

100 mg/kg/d

Group 2

300 mg/kg/d

Group 3

1000 mg/kg/d

HCD

(range)

Postimplantation loss

Mean %

8.9

8.2

7.1

23.1*

12.1

(4.6 – 50.0)

Total resorptions

Mean

0.6

0.6

0.5

1.6*

0.7

(0.4 – 3.1)

Mean %

8.9

8.2

7.1

23.1*

12.0

(4.6 – 50.0)

Late resorptions

Mean

0.2

0.3

0.3

0.9*

---

Mean %

2.0

4.5

3.9

14.2*

---

HCD = Historical control data; * = p ≤ 0.05 (DUNNETT-test [two-sided])

 

 

Table: Occurence of statistically significantly increased fetal skeletal variation (expressed as mean percentage of affected fetuses/litter)

Finding

Group 0

0 mg/kg/d

Group 1

100 mg/kg/d

Group 2

300 mg/kg/d

Group 3

1000 mg/kg/d

HCD

(range)

Incomplete ossification of parietal; unchanged cartilage

0.0

1.1

1.1

4.2*

0.2

(0.0 – 1.0)

Splitting of skull bone

1.2

1.2

2.6

8.1**

2.8

(0.0 – 7.1)

Supemumerary 13th rib; cartilage present

3.8

5.3

2.5

13.1*

4.0

(0.0 – 8.7)

Supemumerary 13th rib; cartilage not present

10.7

10.3

7.6

33.5**

6.2

(2.1 – 13.6)

Total fetal skeletal variations

64.8

63.3

66.4

79.3*

63.1

(46.3 – 78.9)

HCD = Historical control data; * = p ≤ 0.05, ** = p ≤ 0.01 (Wilcoxon-Test [one-sided])

 

 

Table: Total fetal malformations

 

 

Group 0

0 mg/kg/d

Group 1

100 mg/kg/d

Group 2

300 mg/kg/d

Group 3

1000 mg/kg/d

Litter

Fetuses

N

N

25

172

25

165

25

176

16

100

Fetal incidence

N (%)

6 (3.5%)

8 (4.8%)

6 (3.4%)

11 (11%)

Litter incidence

N (%)

6 (24%)

6 (24%)

6 (24%)

7 (44%)

Affected fetuses/litter

Mean%

3.9

4.7

3.2

14.3*

* = p ≤ 0.05 (Wilcoxon-Test [one-sided])

 

 

Table: Total fetal variations

 

 

Group 0

0 mg/kg/d

Group 1

100 mg/kg/d

Group 2

300 mg/kg/d

Group 3

1000 mg/kg/d

Litter

Fetuses

N

N

25

172

25

165

25

176

16

100

Fetal incidence

N (%)

122 (71%)

113 (68%)

125 (71%)

82 (82%)

Litter incidence

N (%)

25 (100%)

24 (96 %)

25 (100 %)

16 (100%)

Affected fetuses/litter

Mean%

71.9

69.7

72.5

83.1*

* = p ≤ 0.05 (Wilcoxon-Test [one-sided]

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP compliant study conducted with the structural analogue substance n-butyl methacrylate
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: IFFA CREDO, Saint Germain sur l'Arbresle, France
- Age at study initiation: sexually mature females, age not specified
- Weight at study initiation: 180-200  grams
- Housing: singly in polycarbonate cages
- Diet (e.g. ad libitum): UAR, Villemoisson, France
- Water (e.g. ad libitum): filtered tap water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 10
- Air changes (per hr): no data
- 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:
Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow (6±20 m3/h). 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%. Food and water were withheld during exposures.
The system used for vapor generation consisted in delivering a constant rate of liquid chemical with an infusion pump at the top of a heated glass column filled with glass beads. Compressed air heated by a glass heater was introduced at the bottom of the glass column in a countercurrent fashion to the liquid flow , an additional air flow rate was passed through the fritted disk of a heated bubbler containing the test chemical. The vaporized compounds were introduced into the main air-inlet pipe of the exposure chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations were monitored continuously with a gas chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. In addition, the exposure levels were determined once during each 6-h exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal. The charcoal samples were then desorbed with carbon disulide. The resulting samples were then analyzed by gas chromatography using appropriate interval standards.
Details on mating procedure:
Alter 2 weeks of acclimatization, nulliparous female rats were housed overnight with adult males (1 male:2 or 3 females) from the same strain and supplier. The day that vaginal smears were found to be sperm-positive was considered day 0 of gestation (GD). Mated females were randomly assigned to treatment groups using a randomization system stratified by body weight on GD 0
Duration of treatment / exposure:
days 6 - 20 of gestation
Frequency of treatment:
6 hours per day
Duration of test:
On GD 21, the females were sacrificed
Remarks:
Doses / Concentrations:
0, 100, 300, 600 or 1200 ppm
Basis:
other: target concentrations (equivalent to 0, 600, 1800, 3600 or 7200 mg/m³)
Remarks:
Doses / Concentrations:
0, 99.6 +/- 5.0, 301.6 +/- 12.2, 602.3 +/-38.0, 1206.4 +/- 46.9 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
22-25 pregnant females per dose
Control animals:
other: yes, concurrently to filtered air
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were recorded on GDs 0, 6, 13, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured for the intervals GDs 6-13 and 13-21

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: no data
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
- Number of death and live fetuses: Yes
Fetal examinations:
- External examinations: Yes: All live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity
- Soft tissue examinations: Yes: Half of the live fetuses from each lifter were preserved in Bouin's solution and examined for internai soft-tissue changes (Barrow and Taylor, 1969; Wilson, 1965).
- Skeletal examinations: Yes: Half of the live fetuses from each lifte were fixed in ethanol (70%), eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination (Staples and Schnell, 1964).
- Head examinations: No data
Statistics:
The number of CL, implantation sites, and live  fetuses, maternal food consumption and various body weights were analyzed  by ANOVA, followed by Dunnett'st-test. the percentage of non-live  implant, resorptions,and males and the proportion of fetuses with  alterations ineach litter were evaluated by Kruskal-Walles test followed  by Dixon-Massey test. Rates of pregnancy and percentage of litters with  any malformations or external, visceral, or skeletal variations were  analyzed using Fisher's test. Where appropriate, least squares analysis  was performed. The level of significance was p < 0.05.
Indices:
FERTILITY AND REPRODUCTIVE PERFORMANCE: The following data were recorded  for each group of numbers of CL, and implantation sites 
- number of   resorptions and viable and dead fetuses. 
- mean fetal body weights 
- fetuses examined for gross malformations and skeletal abnormalities of  sex and of fetuses.
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No test dam died. Exposure to 300 ppm or higher concentrations of n-butyl methacrylate led to a significant decrease in maternal body weight gain during the first half of exposure (Table 1). The overall maternal weight gain during GDs 6-21 and the absolute weight gain were significantly less than control at 1200 ppm. A slight reduction in maternal food consumption was observed during the first half of exposure at 300 ppm and higher concentrations (p < 0.05 and p <0.01 at 300 and 1200 ppm, respectively). No adverse effects on the average number of implantations and live fetuses, incidence of non-live implants or resorptions, or fetal sex ratio were noted among litters exposed to n-butyl methacrylate (Table 2).
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal body weight was significantly reduced at 600 ppm (females) and at 1200 ppm (all, male and female fetuses). These decreases amounted to 5% of the concurrent control values at 1200 ppm. Occasional visceral malformations occurred in low frequency and were distributed across the different groups, including control (Table 3). There were no significant differences between the control and treated groups in the incidences of either individual or total external and visceral variations, or of individual skeletal variations. The only statistically significant changes were higher mean percentages of fetuses with skeletal variations or any variations at the highest concentration of n-butyl methacrylate, compared with the concurrent control. The biological relevance of these findings is limited by the fact that the observed incidences occurred with no clear concentration-related pattern. These increases might be considered at most as slight fetotoxicity.
Abnormalities:
not specified
Developmental effects observed:
not specified

TABLE 1

Change in Weight During Gestation in Sprague-Dawley Rats Inhaling n-Butyl Methacrylate on Days 6 to 20 of Gestation and Euthanized on Day 21

Concentration

ppm/6 h/day

No. of damsa

Body weight (g)

on GD 6

Body weightgain (g) on GD

Absolute

weight gain (g)b

6±13

13±21

6±21

0

24

257 ± 15

33 ± 6

100± 16

133 ± 19

27 ± 10

100

24

259 ± 16

31 ± 8

100± 15

132 ± 18

26 ± 8

300

25

258 ± 15

27 ±6**

106± 12

132 ± 14

25 ± 9

600

22

264 ± 18

26 ± 5**

102± 19

128 ± 19

21 ± 10

1200

25

261 ± 18

21 ± 6**

98 ± 13

119 ± 15*

19 ± 9**

Note.Values are expressed as means ± SD.

a Includes all dams pregnant at euthanization.

bDay 21 body weight) 2 (gravid uterus weight) 2 (Day 6 body weight).

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

TABLE 2
Reproductive Parameters in Sprague-Dawley Rats Inhaling Methacrylic Acid, Ethyl Methacrylate, n-Butyl Methacrylate, or Allyl Methacrylate
on Days 6 to 20 of Gestation and Euthanized on Day 21

Concentration (ppm/6 h/day)

Test animals (dams)

Litters with implants

Litters with live fetuses

 

No. dead/ no. treated

% of females pregnant at euthanization

No. of litters

No. of corpora lutes per dam

No. of implantation sites per litter

% of nonlive implants per littera

% of resorption sites per litter

No. of litters

No. of live

fetuses per

litter

% of males per litter

Average fetal body weight (g) per litter

All

Males

Females

 

0

0/26

92.3

24

15.63 ± 1.69

14.63 ±2.43

7.12 ± 12.12

6.52 ± 9.66

24

13.75 ± 3.01

53.40 ± 16.29

5.70±0.26

5.84 ± 0.29

5.54 ± 0.25

 

100

0/26

92.3

24

15.75 ± 1.45

14.71 ± 1.90

4.00 ± 6.80

4.00 ± 6.80

24

14.13 ± 2.11

51.50 ± 15.16

5.59± 0.25

5.71 ± 0.28

5.47 ± 0.27

 

300

0/26

96.2

25

16.12 ± 1.56

15.24 ±   1.59

4.05 ± 3.89

4.05 ± 3.89

25

14.60 ± 1.38

52.83 ± 12.70

5.53± 0.24

5.67 ± 0.26

5.38 ± 0.20

 

600

0/26

84.6

22

16.27 ± 1.52

15.18 ±2.54

5.28 ±8.08

5.28 ± 8.08

22

14.50 ± 3.02

46.59 ± 11.86

5.51± 0.33

5.72 ± 0.36

5.33 ± 0.35*

 

1200

0/27

92.6

25

16.76 ± 3.30

15.08 ± 2.00

6.27 ± 6.08

6.27 ± 6.08

25

14.12 ± 1.99

46.22 ± 15.16

5.40± 0.24**

5.56 ± 0.29**

5.26 ± 0.24**

 

aResorptions plus dead fetuses.

Values are expressed as means±SD.

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

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
CLP and guidelien compliant study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 800 mg/m³
Study duration:
subacute
Species:
rat
Additional information

Studies of developmental toxicity of t-BMA are not available. Reliable studies of analogous substances were used to assess the potential of t-BMA for developmental toxicity.

Methyl methacrylate and n-butyl methacrylate have been tested in reliable developmental toxicity studies in rats and rabbits.

 

Oral

In a study performed according to OECD guideline 414 and in compliance with GLP, n-butyl methacrylate was tested for its prenatal developmental toxicity in Himalayan rabbits (BASF SE, 2009). The test substance

was administered as an aqueous preparation to 25 time-mated female Himalayan rabbits by stomach tube at doses of 0, 100; 300 and 1000 mg/kg body weight/day on gestation days (GD) 6 through 28.

At terminal sacrifice on GD 29, 18-25 females per group had implantation sites. In the does treated at 1000 mg/kg body weight/day, distinct and statistically significant reduction of food consumption (-44%) and gross and net body weight gain(-47% and -54%, respectively) was recorded. Abortions (and subsequent sacrifice) were observed in 7 of 25 animals. Stomach erosions in 7/25 does, no feces in the small intestine in 5/25 does and watery feces in the intestines in 3/25 high-dose does (stomach erosions and subsequent massive decrease of individual food consumption particularly affected all does with abortions), complete postimplantation loss in 2 individual does secondary to distinct maternal toxicity and a statistically significant decrease of mean gravid uterine weights were observed at necropsy. A statistically significant decrease of mean weights, a slightly higher rate of malformations per litter (but no specific pattern of malformations), treatment-related adverse findings primarily limited to severely fused sternebrae (bony plate) and a slightly higher rate of variations per litter (skeletal variations such as delayed ossification and supernumerary ribs, commonly associated with decreased fetal weight and maternal stress) were observed in fetuses.

At 300 mg/kg body weight/day, does presented a statistically significant reduction of food consumption (-18%) and net (-37%) body weight gain and no biologically relevant differences between treated and control animals were observed in fetuses.

At 100 mg/kg body weight/day, no biologically relevant differences between treated and control animals were observed in does and fetuses. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/d based on reduced food consumption and body weight gain in the dose at 300 mg/kg bw/d and above. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg bw/d based on abortions, decreased fetal growth and bone alterations at 1000 mg/kg bw/d. There were no adverse fetal findings evident at a dose not producing maternal toxicity. The compound is no selective teratogen.

Methyl Methacrylate was tested for its prenatal developmental toxicity after oral application in Himalayan rabbits according to OECD TG 414 in compliance with GLP (BASF SE, 2009). The test substance was administered as an aqueous preparation to 25 inseminated female Himalayan rabbits by stomach tube at doses of 50, 150 and 450 mg/kg body weight/day on GD 6 through GD 28. The control group, consisting of 25 females, was dosed with the vehicle (1% Carboxymethylcellulose CB 30.000 in drinking water and a few drops Cremophor EL and one drop hydrochloric acid [1% CMC]) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 24-25 females per group had implantation sites.

In the mid and high dose group, reduced food consumption (-18% and -13%, resp.) and body weight gain (-31% and -27%, resp.) were noted. No test substance-related adverse effects were observed on gestational parameters or fetuses. In the low dose group, no test substance-related adverse effects on does, gestational parameters or fetuses were observed.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 50 mg/kg bw/d. The NOAEL for prenatal developmental toxicity is ≥ 450 mg/kg bw/d. No adverse fetal findings of toxicological relevance were evident at any dose

Inhalation

In a study comparable to the OECD guideline 414, groups of 22-25 pregnant female rats were given whole-body inhalation exposures to n-butyl methacrylate at target concentrations of 0, 100, 300, 600 or 1200 ppm (analytical concentrations: 0, 99.6 +/- 5.0, 301.6 +/- 12.2, 602.3 +/-38.0, 1206.4 +/- 46.9 ppm) for 6 hr/day, during days 6 to 20 of gestation (GD) (Saillenfait et al., 1999). Maternal toxicity (decreased body weight gain) was shown at 300 to 1200 ppm. Feed consumption was decreased at 1200 ppm. No dam died during the test and there were no adverse effects on the average number of implantations and live fetuses, incidence of non-live fetuses, or on resorptions. Fetal body weights of male pups were significantly reduced at 1200 ppm, and females at 600 and 1200 ppm n-butyl methacrylate. There were no significant differences between control and treated groups for external, visceral, or skeletal malformations. A significant increase in the skeletal variations per litter occurred at 1200 ppm n-butyl methacrylate, compared to controls. The authors concluded that NOAEC for developmental toxicity was 300 ppm n-butyl methacrylate. There was no evidence of embryolethality or teratogenicity with n-butyl methacrylate.

In a developmental toxicity study according to OECD 414 and conducted in compliance with GLP standards (Solomon et al. 1991), methyl methacrylate (99.9% active ingredient) was administered by inhalation exposure to 5 groups (27 rats/group) of presumed pregnant rats (Crl:CDBR) at concentrations of ca. 0 (control), 0.4, 1.2, 4.8, 8.3 mg/L (corresponding to 99, 304, 1178, and 2028 ppm) for 6 hr/day on days 6-15 of gestation (GD) . All doses were administered by a whole-body inhalation exposure under dynamic conditions. Clinical signs were recorded daily on GD 0-20. The dams were weighed on GD 0, 6, 8, 10, 13, 16 and 20. Feed consumption was recorded during gestation. On GD 20, the dams were euthanized and the thoracic and abdominal cavities were examined for gross changes. Each uterus was weighed and corpora lutea, implantation sites and resorptions were counted. Fetuses were weighed, sexed, examined for external alterations and one-half of the fetuses from each litter were examined for visceral alterations.

No treatment-related deaths were noted at any concentration tested. The only clinical sign noted was a minimal increase in the incidence of scant feces at ca. 8.3 mg/L. At all exposure levels tested losses in maternal body weight or decreases in maternal body weight gain and decreases in maternal feed consumption were noted. Loss in maternal body weight during the first two days of exposure followed by an overall reduced increase in maternal body weight gain during the treatment period was detected for the 4.8 mg/L and 8.3 mg/L groups. Slight effects were observed for the 0.4 and 1.2 mg/L treatment groups as indicated by a transiently (during the first two days of exposure) reduced maternal body weight gain. According to the authors, a maternal no observed effect level (NOEL) could therefore not be demonstrated. No embryo of fetal toxicity was evident and no increase in the incidence in the malformations or variations was noted at exposure levels up to and including 8.3 mg/L. Therefore toxicity to the conceptus was not evident even at exposure levels that resulted in overt maternal toxicity.


Justification for selection of Effect on developmental toxicity: via oral route:
Most suitable study

Justification for selection of Effect on developmental toxicity: via inhalation route:
Most suitable study

Justification for classification or non-classification

In several reliable studies of the analogous substances methyl methacrylate and n-butyl methacrylate in rats and rabbits, no effects on fertility or developmental toxicity were observed, even in maternal toxic doses. Therefore, based on the available experimental data on the read-across substances, the test item is not classified and labelled for reproduction/fertility and developmental toxicity according to Regulation (EC) No 1272/2008 (CLP) and Directive 67/548/EEC.