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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
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
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
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).
Remarks:
Doses / Concentrations:
0, 50, 150, 400 mg/kg/day
Basis:
nominal conc.
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.
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: adverse effects on food consumption observed at the LOAEL of 150 mg/kg bw/day in the F0 parental females
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
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment- related effects
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:
GLP and guideline study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A screening study according to OECD test guideline 422 was performed to assess the potential of ureido methacrylate for toxicity on reproduction in rats. Additionally, a 2-generation study according to OECD test guideline 416 with rats is available which studied effects of the analogous substance methyl methacrylate (CAS-No. 80-62-6).

oral

The possible toxic effects, including screening of the toxicity on reproduction, of the test substance, ureido methacrylate (purity unknown), were evaluated in a GLP conform combined repeated dose study with a Reproduction/ Developmental screening test in rats following OECD test guideline 422 (Rohm & Haas 2002). The test substance was administered orally by gavage to four groups of 12 male and 12 female Crl:CD BR rats once daily for eight weeks. Dose levels were 0, 100, 300, and 1000 mg a.i./kg/day, administered at a dosage volume of 5 ml/kg. The control group received the vehicle, double-distilled water, on a comparable regimen at a dosage volume of 5 ml/kg. Males and females of the same treatment group were mated 1:1 two weeks after the beginning of the treatment.

During the treatment period, each rat was observed twice daily for morbidity or mortality. General clinical observations were made at least once daily. Body weight was determined weekly for males throughout this study. Females were weighed weekly until cohabitation, on Gestation Days (G) 0, 3, 6, 9, 12, 15, 18, and 20, and on Postnatal Days (PND) 0 and 4. Food consumption was determined weekly for male and female animals until cohabitation. Food consumption was not measured for either sex during cohabitation due to the inability to ascribe consumption to individual animals. Females feed consumption was measured from G0-7, G7-14, G14-21 and from PND 0-4 during lactation. The weekly feed consumption was resumed for males after the mating period and continued throughout the study. On Postnatal Day 0 all offspring were examined externally, and their status (e.g., alive, dead, stillborn, or uncertain), sex, and weight were determined. On Post Natal Day 4 pups were again counted, sexed, weighed, and examined for gross abnormalities prior to being euthanized.

Pups were discarded without further evaluation.After eight weeks, overnight fasted animals were euthanized and necropsied. Hematology and clinical chemistry measurements were performed on all animals at terminal necropsy. Animals were fasted overnight and blood samples were collected just prior to terminal necropsy. Microscopic examination of the studied organs were performed for 5 (randomly selected) parental animals/sex in the high dose group and control group. All tissues exhibiting gross pathological changes were examined microscopically.

Treatment-related decreases in hemoglobin (6%), hematocrit (7%), and mean cell volume (4%) were noted in males at 1000 mg a.i./kg bw/day. In addition, platelet counts were increased (14%) at this level. Treatment-related increases in absolute and relative kidney weights (10-11%) and in absolute and relative liver weights (20%) were noted in both sexes at 1000 mg a.i./kg. Absolute and relative liver weights were also increased (12-16%) in males at 300 mg a.i./kg. Other effects were considered as incidental and not treatment-related. Microscopic examinations in the relevant organs revealed no complementary effects seen in liver and kidney weights, these observations are considered as not adverse.

There were no treatment related effects on the studied reproduction parameters (precoital interval, duration of gestation, gestation index, changes in lactation, estrus cycle and sperm, number of implantations and corpora lutea, ovarian primordial follicle counts, litter size and weights, sex ratios, viability index and postnatal survival until weaning).

Based on the results of this study, the NOEL (no-observed-effect level) for systemic toxicity of ureido methacrylate administered orally is 100 mg a.i./ kg bw/day in males and 300 mg a.i./ kg bw/day in females. The NOAEL for toxicity on reproduction is assessed to be >= 1000 mg a.i. /kg bw/ day.

Additionally, data were adopted by read-across from a reliable two-generation reproduction toxicity study of methyl methacrylate (CAS 82-60-6). Due to the structural similarities, the same results can be expected for ureido methacrylate).

The study was performed in rats with oral administration (gavage) according to OECD 416 in compliance with GLP (REACH Methyacrylate Task Force, 2009). In this study, 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).

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.


Short description of key information:
oral
Reproduction/Developmental Toxicity Screening Test (rat, oral, gavage): NOEL parental toxicity: 100 mg/kg bw/day in males and 300 mg/kg bw/day in females (due to increased organ weights); NOAEL fertility >= 1000 mg/kg bw/day (no effects observed; GLP, Rohm & Haas 2002)

Analogous substance (read across): Methyl methacrylate (CAS No. 80-62-6)
Two-generation reproduction toxicity study in rats (OECD TG 416): NOAEL general, systemic toxicity: 50 mg/kg bw/day (due to adverse effects on food consumption); NOAEL fertility and reproductive performance: 400 mg/kg bw/ day (GLP, REACH Methacrylate Task Force, 2009).

Justification for selection of Effect on fertility via oral route:
Only one GLP and guideline study available.

Effects on developmental toxicity

Description of key information
oral
Reproduction/Developmental Toxicity Screening Test (rat, oral, gavage): NOEL parental toxicity: 100 mg/kg bw/day in males and 300 mg/kg bw/day in females (due to increased organ weights); NOAEL embryotoxicity/teratogenicity >= 1000 mg/kg bw/day (no effects observed; GLP, Rohm & Haas 2002)
Prenatal Developmental Toxicity Study (rabbit, oral, gavage), Read across: Methyl Methacrylate, CAS 80-62-6: NOAEL (maternal toxicity): 50 mg/kg bw/d, NOAEL (prenatal developmental toxicity): 450 mg/kg bw/d (highest dose tested)
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-10-23 to 2000-12-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh Facility, Raleigh, NC)
- Age at study initiation: 11 weeks
- Weight at study initiation: males weighed 357.5 - 358.1 g; and females weighed 240.4 - 241.3 g
- Fasting period before study:
- Housing: Animals were individually housed in stainless steel cages (34 cm x 18 cm x 18 cm) with wire-mesh fronts and bottoms, suspended over pans containing absorbent liners. Cage liners were changed at least three times weekly. During mating, cage liners were changed daily. Clean cage banks were supplied approximately every two weeks. After mating, females were housed in polycarbonate shoe-box cages (53 cm x 29 cm x 20 cm) containing Alpha-Dri bedding (Shepherd Specialty Papers, Irre.) throughout gestation and lactation.
- Diet: During the acclimation and treatment periods, all animals had free access to PMI Certified Rodent Diet #5002M (Ralston Purina Co., St. Louis, MO).
- Water: Water purified by reverse osmosis was available ad libitum via an automatie watering system or water bottles.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 41-63
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: Reverse Osmosis (RO) Water
Details on exposure:
Dosage: 5 mL/kg bw
The appropriate amount of test substance was used to prepare sufficient quantities of gavage solutions at concentrations appropriate to deliver dose levels of 100, 300, and 1000 mg ai/kg/dayat a constant volume of 5 mL/kg. The dose levels were adjusted to represent percent active ingredient. The test substance was weighed in a hood and diluted in reverse osmosis (RG) water. Group 1 (control) received RO water only. Gavage solutions were prepared once a week and stored in the refrigerator. Prior to gavaging the animals each day, an aliquot of each concentration was placed in a small beaker and allowed to warm to approximate room temperature. Daily dosing solutions and any treated gavage solution unused at the end of a dosing period were discarded as hazardous waste.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance in RO water, when stored refrigerated for 8 days, was determined from the first dose preparation. Homogeneity was
determined by analyzing top, middle and bottom sampIes from the first dose preparation. SampIes of dosing solutions prepared on days 34 and 52 were analyzed for active ingredient content to determine proximity to target concentrations.
The sampIes were diluted to 100 ppm with acetonitrile and analyzed by HPLC. Proximity to target for the sampIes analyzed ranged from 89.3% to 100.0%. Day 8 stability of the sampIes was confirmed. Homogeneity analysis was done by performing proximity to target far the top, middle, bottom sampIe and reporting the standard deviation. The standard deviation ranged from 1.19 to 3.39 (n = 3). There were no issues of homogeneity.
Details on mating procedure:
Exposure of parental animals (12/sex/dose) began when the animals were approximately eleven weeks of age. Adult female rats were placed individually with an assigned male from the same treatment group, and observed daily until copulation was verified by the presence of a sperm plug in situ, multiple sperm plugs on the cage liner (at least 3), and/or sperm in a vaginal lavage sample. The day that positive evidence of mating was observed was designated as Day 0 of gestation, or GD0.
Animals were paired for a maximum period of 2 weeks. Females that showed no evidence of copulation were assumed pregnant at the end of the mating period, and housed accordingly.
Duration of treatment / exposure:
8 weeks
Frequency of treatment:
daily
Duration of test:
8 weeks
Remarks:
Doses / Concentrations:
100, 300, 1000 mg a.i./kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The test substance was administered daily via gavage at dose levels of 0 (control), 100, 300, or 1000 mg a.i./kg of body weight/day (Groups 1, 2, 3, and 4, respectively). The limit dose of 1000 mg a.i./kg bw/day was selected based on agavage range-finding study which induced minimal toxicity in the parameters measured. The low and middle doses were approximately logarithmically spaced from the high dose.
- Rationale for selecting satellite groups: no satellite groups
Maternal examinations:
PARAMETERS ASSESSED DURING STUDY P AND F1:
- Clinical observations: Body weight, feed consumption and clinical signs were monitored in parental animals throughout treatment.
Ovaries and uterine content:
No data
Fetal examinations:
On Postnatal Day 0 all offspring were examined externally, and their status (e.g., alive, dead, stillborn, or uncertain), sex, and weight were determined. On Post Natal Day 4 pups were again counted, sexed, weighed, and examined for gross abnormalities prior to being euthanized. Pups were discarded without further evaluation.
Statistics:
The litter (i.e. proportion of pups/litter, or litter mean) was used, where appropriate, as evaluation. The level of statistical significance selected was p<0.05, unless otherwise noted. The statistical tests that were used to analyze the parameters studied are listed below.
Analysis of Variance (ANOVA): Parental Body Weight; Parental Body Weight Gain; Parental Feed Consumption; Parental Water Consumption; Duration of Gestation; Number of Days Until Mating; Pups Delivered (Total); Live Pups/Litter; Pup Weight/Litter; Implantations; Corpora Lutea

2 x N Chi-square test: Male and Female Mating Index; Male and Female Fertility Index; Females Gestation Index; Females Completing Delivery Index; Females with Stillborn Pups; Females with all Stillborn Pups; Days 'N' to 'N' Mating; Litters with Liveborn, but No Pups Alive; Live Birth Index; Stillborn Pups Dying, Missing and/or Cannibalized; Viability Index (pups surviving 4 days); Sex Ratio

2 x N Kruskal-Wallis nonparametric ANOVA: Pre- & Post-Implantation Loss
Indices:
Male and Female Mating Index; Male and Female Fertility Index; Females Gestation Index; Females Completing Delivery Index; Live Birth Index; Viability Index (pups surviving 4 days); Sex Ratio
Historical control data:
no data
Details on maternal toxic effects:
Details on maternal toxic effects:
- Mortality and time to death: There were no treatment-related deaths were noted in female parental animals during premating, gestation or lactation, in pups during lactation, or in males throughout the study at dose levels up to including 1000 mg a.i./kg bw/day. One female at 300 mg a.i./kg bw/day was euthanized in a moribund condition. This death was not considered treatment-related but due to dosing error. Microscopic evaluation confirmed inadvertent dosing trauma.
- Clinical signs: No treatment-related effects were seen in the Detailed Clinical Observations or Functional Observational Battery parameters. There were no treatment-related effects noted in motor activity in either sex up to and including 1000 mg a.i./kg bw/day.
- Body weight gain: There were no treatment-related effects on body weight or feed consumption in females during the premating, gestation or lactation periods or in males throughout the study at dose levels up to and including 1000 mg a.i./kg bw/day.
- Food/water consumption: There were no treatment-related effects on feed consumption in females during the premating, gestation or lactation periods or in males throughout the study.
- Precoital interval: There were no treatment-related effects at any dose level.
- Duration of gestation: There were no treatment-related effect on gestation index (percent of pregnant females that delivered live offspring) which was 100% for all doses.
- Gestation index: There were no treatment-related effects.
- Changes in lactation: There were no treatment-related effects on offspring viability during lactation in any treatment groups.
- Changes in estrus cycles: There were no treatment-related effects.
- Number of implantations: There were no effects on the number of implantation sites or in post-implantation loss at any dose level.
- Number of corpora lutea: There were no treatment-related effects.
- Ovarian primordial follicle counts: There were no treatment-related effects.
- Organ weights: There were no treatment-related effects on absolute or relative organ weights in females at concentrations up to and including 300 mg a.i./kg. Treatment-related increases in absolute and relative kidney weights (10-11%) and in absolute and relative liver weights (20%) were noted at 1000 mg a.i./kg bw/day. There was not a statistically significant change in relative epididymus weight and there were no corresponding histopathologic findings in the epididymus or in the prostrate or testes. In addition, there were no adverse reproductive effects noted at this level. A statistically significant increase in relative spleen weight in females at 100 mg a.i./kg bw/day was considered incidental since similar effects were not seen at higher levels.
- Histopathology incidence and severity: There were no treatment-related gross findings in maternal animals at any dose level.
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
- Litter size and weights: There were no treatment-related effects on number of pups per litter in any treatment group. There were no effects on pup body weight at any dose.
- Number viable (number alive and number dead): There were no treatment-related effects on offspring viability during lactation in any treatment group.
- Sex ratio: There were no treatment-related effects on the ratio of male to female pups in any treatment group.
- Grossly visible abnormalities, external, soft tissue and skeletal abnormalities: There were no external abnormalities noted.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and guideline study.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A screening study according to OECD test guideline 422 was performed to assess the potential of ureido methyl methacrylate for toxicity on reproduction in rats. Additionally, a developmental study according to OECD test guideline 414 with rabbits is available which studied effects of the analogous substance methyl methacrylate (CAS-No. 80-62-6).

oral

The possible effects on development of ureido methyl methacrylate were evaluated in the GLP conform combined repeated dose study with a Reproduction/ Developmental screening test in rats following OECD test guideline 407 as described above (Rohm & Haas 2002).

There were no treatment related effects on the studied reproduction parameters. Based on the results of this study, the NOEL (no-observed-effect level) for systemic toxicity of ureido methyl methacrylate administered orally is 100 mg a.i./ kg bw/day in males and 300 mg a.i./ kg bw/day in females. The NOAEL for toxicity on development is assessed to be >= 1000 mg a.i. /kg bw/ day.

 

Additionally, methyl methacrylate (CAS 82-60-6), a structural similar substance, was tested for its prenatal developmental toxicity after oral application in Himalayan rabbits according to OECD 414 in compliance with GLP (REACH Methacrylate Task Force, 2009). Due to the structural similarities, the same results can be expected for allyl methacrylate. The test substance was administered 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 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.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one GLP and guideline study available.

Justification for classification or non-classification

Based on the results of a screening test with ureido methacrylate and a 2 -generation study and a developmental toxicity study with the analogous substance methyl methacrylate, there is no indication given to classify ureido methacrylate for its reprotoxic potential according to 67/548/EEC and Regulation (EC) No 1272/2008 (GHS, CLP), respectively.

Additional information