Methacrylic anhydride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data available for MAAH.

Read across data from methacrylic acid, MAA (hydrolysis product and primary metabolite; screening level) and methyl methacrylate, MMA (metabolite donor substance; read across justification see attached document):

2-gen reproduction study, OECD 416, oral, rat, MMA: NOEL P/F1 systemic 50 mg/kg bw/d (bw gain; food consumption); NOAEL P/F1 fertility 400 mg/kg bw/d (no effects seen)

90 d repeated dose, OECD 413, inhal, rat, MAA: NOAEC systemic 100 ppm (bw gain, food consumption); NOAEC sexual organs 350 ppm (no effects seen)

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

GLP compliance:

yes (incl. QA statement)

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:

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:

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 2

Endpoint:

toxicity to reproduction

Remarks:

other: 90 d inhalation study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline inhalation study with additional hisopathology of reproductive organs and sperm investigations

Guideline:

other: OECD 413, 90 d inhalation study

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age when supplied; sex: about 7 weeks, male and female
Supplier: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
During the period when the rats were not exposed they were housed singly in wire cages (type DK III, Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm²)). Underneath the cages, waste trays were fixed containing bedding material (Type Lignocel FS14 fibres,
dustfree bedding supplied by SSNIFF, Soest, Germany)
The motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, FRG (floor area about 800 cm²) and bedding.
The animals were kept in fully air-conditioned rooms in which a temperature in the range of 20 - 24°C and relative humidity in the range of 30 - 70% were ensured by means of a central air-conditioning system.
A light/dark rhythm of 12 hours was maintained.
The room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalinammonia-based terminal disinfector) before the start of the study. Usually, each week the floor and the walls were cleaned with water containing about 1 % Mikroquat®.
The animals were maintained on milled mouse/rat laboratory diet “GLP”, (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) and tap water ad libitum.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer at a constant rate by means of the piston metering pump. The vapor was generated by spraying the substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres in test groups 1 - 4 were analyzed by HPLC.

Duration of treatment / exposure:

90 d

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
0, 70 mg/m³ (20 ppm), 141 mg/m³ (40 ppm), 352 mg/m³ (100 ppm ), 1232 mg/m³ (350 ppm )
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Details on study design:

In the morning, blood was taken from the retro-orbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence. At necropsy specimen were sampled from fasted anesthetized male animals in a randomized sequence for sperm analyses.
Hematological parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters.
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semi-quantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.
Sperm motility examinations were carried out in a randomized sequence. Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest dose group, only.
The animals were killed under Narcoren anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were then be necropsied and subjected to a grosspathological assessment. Animals that died intercurrently or were killed in a moribund state were necropsied and assessed by gross-pathology as quickly as possible.

Sperm parameters (parental animals):

At necropsy specimen were sampled from fasted anesthetized male animals in a randomized sequence for sperm analyses.
Sperm motility examinations were carried out in a randomized sequence. Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest dose group, only.

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive effects observed:

not specified

Conclusions:

In a 90 d inhalation study with MAA histopathology of the sexual organs and additional fertility parameters were investigated. Up to 350 ppm (1232 mgf/m³) no effects were seen on gross pathology including organ weights, histopathology, sperm motility or sperm morphology.

Executive summary:

In a 90 d inhalation study according OECD 413 with methacrylic acid histopathology of the sexual organs and additional fertility parameters were investigated. Up to 350 ppm (1232 mg/m³) no effects were seen on gross pathology including organ weights, histopathology, sperm mobility or sperm morphology. Under the current test conditions, the NOAEC for fertility related parameters was 350 ppm (1253 mg/m3) for male and female rats.

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no studies available for reproductive toxicity of methacrylic anhydride. As methacrylic anhydride was demonstrated to hydrolyse very fast into methacrylic acid, and methacrylic acid is the hydrolysis product and primary metabolite of methacrylic anhydride, this endpoint is covered by read across to data of methacrylic acid (MAA) or methyl methacrylate (MMA), the metabolite donor substance (see attached read across justification).

In a OECD 413 90-day inhalation study 10 male and 10 female Sprague Dawley rats were whole body exposed to target concentrations of 20, 40, 100 and 350 ppm (corresponding to 72, 143, 358 and 1253 mg/m3) methacrylic acid for 6 hrs/d, 5 days/wk for 90 days (65 exposures; BASF 2008). In addition to examination of the standard organs and tissues, the sexual organs and parameters of fertility were examined. These examinations included weighing of sexual organs in both males and females and sperm mobility, morphology and sperm head count in one testis and one epididymis of each male animal of the high concentration and control group. Substance-related changes of the sexual organs were not noted in any of the exposed animals (males or females), nor were there any changes of sperm mobility and sperm head counts. Under the current test conditions, the NOAEC for fertility related parameters was 350 ppm (1253 mg/m3) for male and female rats.

While there is no full reproduction study with methacrylic acid, methyl methacrylate has 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, 2009a)

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).

 

Human data

No reliable data available

Effects on developmental toxicity

Description of key information

No data available for MAAH.

Read across data from methacrylic acid, MAA (hydrolysis product and primary metabolite) and methyl methacrylate, MMA (metabolite donor substance; read across justification see attached document):

OECD 414, inhal, rat, MAA: NOAEC systemic 200 ppm (bw gain, food consumption); NOAEC developmental 300 ppm (no effects seen)

OECD 414, oral, rabbit, MMA: NOEL systemic 50 mg/kg bw/d (food consumption); NOAEC developmental 450 mg/kg bw/d (no effects seen)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

DATA QUALITY: Study was conducted in accordance with a recognized scientific procedure for determining the developmental toxicity of a test substance when administered repeatedly via inhalation. Study was conducted incompliance with GLP regulations. The study meets national and international scientific standards (OECD 414) and provides sufficient information to support the conclusions regarding the NOAEL and the LOAEL demonstrated from the study data.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Nulliparous female Sprague-Dawley rats, weighing 180-200  grams.obtained from IFFA Credo Breeding Labs. AGE at Start of Test: sexually mature females; age not specified. Mated females were  housed inclear polycarbonate cages with stainless steel wire lids and  hardwood shavings for bedding. Food and water available adlibitum except  during exposures.

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

 Exposures were whole  body and conducted in a 200 L chamber. Chamber temperature was 23 degrees  C, and the relative humidity was 50%. Air was passed through a heated  bubbler containing test material. The vaporized material was then  introduced into the exposure chambers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

 Concentrations were monitored  continuously with a GC, and were determined once during each 6 hr  exposure by collecting the material and analyzing against a standard  using GC.   

Details on mating procedure:

2-3 females were caged with one male rat for mating. The  onset of gestation was based upon the presence of sperm in the vaginal  smear and this was designated gestation day 0. After confirmation of  mating, females werere turned to an individual cage. 

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

day 6 to 20 of gestation

Duration of test:

Mated females were exposed 6 hr/day on days 6 through  20 of gestation.

Dose / conc.:

50 ppm

Remarks:

corresponds to 179 mg/m3

Dose / conc.:

100 ppm

Remarks:

corresponds to 358 mg/m3

Dose / conc.:

200 ppm

Remarks:

corresponds to 716 mg/m3

Dose / conc.:

300 ppm

Remarks:

corresponds to 1076 mg/m3

No. of animals per sex per dose:

22-25 pregnant females per dose.

Control animals:

other: yes, concurrently to filtered room air

Ovaries and uterine content:

 The uterus was removed and weighed. At necropsy, the uterine horns and ovaries were exposed  to count the C.L., implantation sites, resorption sites, and viable and  dead fetuses.   FERTILITY AND REPRODUCTIVE PERFORMANCE: The following data were recorded  for each group of numbers of CL, and implantation sites o number of  resorptions and viable and dead fetuses. O mean fetal body weights o  fetuses examined for gross malformations and skeletal abnormalities of  sex and of fetuses.

Fetal examinations:

Live fetuses were weighed, sexed, and examined for external  anomalies. 50% of the live fetuses were preserved in Bouin's solutionand  examined for internal soft-tissue changes. The remaining fetuses were  fixed in ethanol (70%), eviscerated and then processed for skeletal  staining with alizarin red S.

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.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Absolute weight gain was significantly reduced at 300 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Fopod consumption was reduced at 300 ppm.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
All animals survived the exposure. Exposure to 300 ppm led to significant decreases in maternal weight gain and food consumption throughout exposure. Absolute weight gain was significantly reduced at 300 ppm.

Dose descriptor:

NOAEL

Effect level:

200 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponds to 716 mg/m³

Abnormalities:

effects observed, treatment-related

Localisation:

other: unspecific (body weight)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no significant changes in number of implantations and live fetuses, in the incidence of non-live implants and sorptions, or in fetal weights across groups. One fetus of 200 ppm and two of the 300 ppm group showed different types of malfomations. There was no consistent pattern of changes to suggest any treatment-related effects. The difference of fetuses with external, visceral, and skeletal variations did not differ between the control and the treated groups. No significant increase in embryo/fetal lethality or fetal malformations were observed after exposure to methacrylic acid. While maternal toxicity was observed, methacrylic acid caused no  evidence of developmental toxicity up to 300 ppm.

Dose descriptor:

NOAEL

Effect level:

>= 300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Remarks on result:

other: corresponds to 1076 mg/m³

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Using a valid scientific method, no significant increase in embryo/fetal lethality or fetal malformations were observed after exposure to methacrylic acid. While maternal toxicity was observed, methacrylic acid caused no  evidence of developmental toxicity up to 300 ppm.

Executive summary:

In an OECD 414 prenatal developmental toxicity study using whole body inhalation methacrylic acid produced no embryo - or foetal lethality, nor fetal malformations after exposure with methacrylic acid, despite overt maternal toxicity (decreased body weight and feed consumption). The NOEC (teratogenicity) was considerd to be 300 ppm (1076 mg/m³).

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 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

Himalayan

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: 16-21 weeks
- Weight at study initiation: 2187-2917 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 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- 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 maximum intervals of 7 days, 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 (conical Erlenmeyer flasks with groundin stopper), topped up (shortly under the marking) with 1% Carboxymethylcellulose solution 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 flask was sealed and the preparation was intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer and the vessels were kept closed between the withdrawals of the preparations.

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:

After an acclimatization period of at least 5 days, the female rabbits were fertilized by means of artificial insemination. This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe (Receptal) were injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study. The day of insemination was designated as gestation day (GD) 0 and the following day as GD 1.

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

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

actual dose: 41 mg/kg bw/d

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

actual dose: 132 mg/kg bw/d

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

actual dose: 406 mg/kg bw/d

No. of animals per sex per dose:

25 inseminated 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 0-29).
- 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 0-29).
- Food consumption: The food consumption was determined daily on GD 1–29.
- Body weight data: All animals were weighed on GD 0, 2, 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). 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 phenobarbital (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.

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the low-, mid- and high-dose rabbits (50; 150 and 450 mg/kg bw/d) were not significantly different from the concurrent control throughout the course of the study. The average body weight gain of the mid- and high-dose rabbits was statistically significantly reduced by about 27% and 31% during the treatment period. A significant reduction of mean body weight gain was also noted for the the high-dose rabbits on GD 19-21.

Corrected (net) body weight gain: Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were comparable among all groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption in the high-dose females (450 mg/kg bw/d) was distinctly and statistically significantly reduced during a significant part of the treatment period (GD 15-23). During the entire treatment period (GD 6-28) the total average food consumption of the high dose rabbits was about 18% below controls. The food consumption of the mid dose females (150 mg/kg bw/d) was similarly affected in terms of magnitude and course of reduction, however the reduction of food consumption reached statistical significance only on GD 22-24. During the treatment period (GD 6-28) the total average food consumption of the mid-dose rabbits was about 13% below controls. Overall, the food consumption of the low-dose does (50 mg/kg bw/d) did not show test substance-related impairments. The reduced food consumption at the 150 and 450 mg/kg bw/d levels is considered to be related to the treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

:At necropsy, only spontaneous findings were seen in single females of every test group. No test substance-related findings were observed in the doses.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate reached 96% in test groups 1 and 3 (50 and 450 mg/kg bw/d) and 100% in test groups 0 and 2 (0 and 150 mg/kg bw/d). Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 24-25 pregnant rabbits per group had implantation sites in the uterus, at terminal sacrifice. There were no test substance-related and/or biologically relevant differences between the control and all dosed 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.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

uterus weight: The mean gravid uterus weights of test groups 1, 2, and 3 (50; 150 or 450 mg/kg bw/d) did not show statistically significant differences in comparison to the control group.

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

other: no adverse effects observed; actual dose 406 mg/kg/d

Dose descriptor:

NOEL

Remarks:

food consumption

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: actual dose: 41 mg/kg/d

Abnormalities:

effects observed, treatment-related

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of all treated groups were not influenced by the test substance. Neither female nor male weights showed statistically significant or biologically relevant differences between the test substance-treated groups and the controls.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (50; 150 and 450 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One sole external malformation (unilateral microphthalmia) was recorded for two fetuses from 2 litters in the high-dose group (450 mg/kg bw/d). This malformation is present in the historical control data. 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.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations of the fetal skeletons were noted in fetuses of test groups 0, 2 and 3 (0; 150 and 450 mg/kg bw/d). Neither statistically significant differences between treated groups and the control were calculated nor a dose-response relationship was observed. All individual skeletal malformations were present in the historical control data at a comparable frequency.
- 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 in the low- and the high-dose groups on a fetus per litter basis. Several specific skeletal variations were statistically significant higher than the concurrent control in the dosed groups (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 and all observed incidences were within the historical control data. Thus an association of these findings to the treatment is not assumed.
- Fetal skeletal unclassified cartilage observations: Additionally, isolated cartilage findings without impact on the respective bony 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 comparable to historical control data and, therefore, regarded to be spontaneous in nature.

Visceral malformations:

no effects observed

Description (incidence and severity):

- 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; 50; 150 and 450 mg/kg bw/d). With the exception of a lateral pouch in the tongue of 2 fetuses all individual soft tissue malformations were present in the historical control data at comparable frequencies. No statistically significant differences between the test groups and the control were observed. The total incidences of external malformations were comparable to the historical control data. 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, hemorrhagic thymus or ovary and blood coagulum around urinary bladder, were recorded for some fetuses of test groups 0, 1, 2 and 3 (0; 50; 150 and 450 mg/kg bw/d). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance induced effect is not assumed.

Details on embryotoxic / teratogenic effects:

- 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. All individual malformations are present in the historical control data, with the exception of lateral pouches in the tongue of 2 fetuses. They did neither show a consistent pattern since a number of morphological structures of different ontogenic origin were affected nor a clear dose-response relationship. The overall incidence of malformations was comparable to the historical control data. 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. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter were not significantly different from the concurrent control and their frequency is comparable to the historical control data. Therefore, they were not considered to be related to the treatment. A spontaneous origin is also assumed for external, soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups including controls (0, 50; 150 and 450 mg/kg bw/d). Distribution and type of these findings do not suggest relation to treatment.

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

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 50 mg/kg/d	Group 2 150 mg/kg/d	Group 3 450 mg/kg/d	HCD (range)
Incomplete ossification of parietal; unchanged cartilage	0.0	0.0	1.9*	2.1*	0.4 (0.0 – 2.6)
Incomplete ossification of hyoid; cartilage present	11.2	11.4	19.1	20.4*	9.8 (0.0 – 21.6)
Splitting of skull bone	0.4	3.3*	3.3	2.3	2.9 (0.0 – 7.7)
Incomplete ossification of cervical centrum; unchanged cartilage	2.5	2.2	3.6	7.3*	2.5 (0.0 – 9.3)
Supemumerary 13th rib; cartilage not present	2.5	9.8	6.1	9.9*	6.6 (0.0 – 17.5)
Total fetal skeletal variations	46.3	63.7*	59.3	71.6**	63.5 (46.3 – 81.9)

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	25 171	24 154	25 157	24 158
Fetal incidence	N (%)	4 (2.3%)	2 (1.3%)	6 (3.8%)	9 (5.7%)
Litter incidence	N (%)	4 (16%)	1 (4.2%)	4 (16%)	7 (29%)
Affected fetuses/litter	Mean%	2.3	1.2	3.6	6.2

 

 

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	25 171	24 154	25 157	24 158
Fetal incidence	N (%)	106 (62%)	106 (69%)	106 (68%)	122 (77%)
Litter incidence	N (%)	21 (84%)	24 (100%)	24 (96%)	23 (96%)
Affected fetuses/litter	Mean%	59.9	69.8	64.3	74.2

 

Conclusions:

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

Executive summary:

The study was performed according to OECD TG 414 in compliance with GLP.

Methyl Methacrylate was tested for its prenatal developmental toxicity in Himalayan rabbits. 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 gestation days (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.

The following test substance-related adverse effects/findings were noted:

Test group 3 (450 mg/kg body weight/day):

-        Reduced food consumption (-18%) and body weight gain (-31%)

-        No test substance-related adverse effects on gestational parameters or fetuses

 

Test group 2 (150 mg/kg body weight/day):

-        Reduced food consumption (-13%) and body weight gain (-27%)

-        No test substance-related adverse effects on gestational parameters or fetuses

 

Test group 1 (50 mg/kg body weight/day):

-        No test substance-related adverse effects on does, gestational parameters or fetuses

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is nominal 450 mg/kg bw/d (actual 406 mg/kg bw/d), the highest dose tested. The no observed effect level (NOEL) for maternal toxicity is nominal 50 mg/kg bw/d (effective 41 mg/kg bw/d) 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 (actual 132 mg/kg bw/d).

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is nominal 450 mg/kg bw/d (actual 406 mg/kg bw/d). No adverse fetal findings of toxicological relevance were evident at any dose.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Study was conducted in accordance with a recognized scientific procedure for determining the developmental toxicity of a test substance when administered repeatedly via inhalation. Study was conducted incompliance with GLP regulations. The study meets national and international scientific standards (OECD 414) and provides sufficient information to support the conclusions regarding the NOAEL and the LOAEL demonstrated from the study data.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no studies available for developmental toxicity of methacrylic anhydride. As methacrylic anhydride was demonstrated to hydrolyse very fast into methacrylic acid, and methacrylic acid is the hydrolysis product and primary metabolite of methacrylic anhydride, this endpoint is covered by read across to data of methacrylic acid (MAA) or methyl methacrylate (MMA), the metabolite donor substance (see attached read across justification).

Non-human data

The OECD SIAR concluded that: “Methacrylic acid (MAA), the common metabolite for all the esters, also was tested in groups of 19-25 pregnant female rats (whole-body inhalation exposure for 6 hr/day, during days 6 to 20 of gestation), at 0, 50, 100, 200, and 300 ppm (0, 179, 358, 716 and 1076 mg/m3) and produced no embryo- or foetal lethality, nor foetal malformations after exposure with MAA at any concentration, despite overt maternal toxicity (decreased body weight and feed consumption) at 300 ppm (1076 mg/m3). The NOAEL for developmental toxicity was considered 300 ppm (1076 mg/m3) MAA (Saillenfait et al., 1999). ”

Methyl methacrylate (MMA) has been tested in a series of developmental toxicity studies on rats and rabbits. In a developmental toxicity study according to OECD 414 MMA was administered by inhalation exposure to 99, 304, 1178, and 2028 ppm (412, 1285, 4900, 8436 mg/m³ (Solomon et al., 1991; currently not selected for the IUCLID dataset). No relevant maternal, treatment-related effects were noted at any concentration tested. No embryo of foetal toxicity was evident and no increase in the incidence in the malformations or variations was noted at exposure levels up to and including 2028 ppm. In this study the NOAEC for developmental toxicity was 2028 ppm (8436 mg/m³).

Since the ESR and the OECD review another study with MMA has been performed, an oral OECD 414 study in rabbits at 50, 150, and 405 mg/kg/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 450 mg/kg bw/d. No adverse foetal findings of toxicological relevance were evident at any dose, even in the presence of maternal toxicity(i.e. NOEL of 50 mg/kg bw/ d based on reduced food consumption).

Human data

No reliable data available.

Justification for classification or non-classification

In analogy of its hydrolysis product and initial metabolite, methacrylic anhydride is not regarded as toxic to reproduction as there was found no specific reproductive toxicity with methacrylic acid.