Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral

Cyclohexyl methacrylate, Modified Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test, rat: NOAEL general, systemic toxicity:300 mg/kg bw/d based on functional liver impairment; NOAEL fertility and reproductive performance: 1000 mg/kg bw/d; NOAEL developmental toxicity: 1000 mg/kg bw/d

Analogous substance methyl methacrylate (CAS# 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; no effects observed (BASF SE, 2010). 

Inhalation 

Analogous substance cyclohexanol (CAS 108-93-0), reproductive screening study in rats: NOAEC general systemic toxicity: 610 mg/m³; based on mortality in high dose group; NOAEC reproductive performance: 610 mg/m³; based on an increased incidence of pregnancies with no viable pups at birth and lower F1 pup body weights (US EPA, 2010).

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-24 to 2017-10-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
yes
Remarks:
Please refer to Principles of Method.
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 Sep 1998
Deviations:
yes
Remarks:
Please refer to Principles of Method.
Principles of method if other than guideline:
The duration of treatment covered a 10 weeks premating period and 2 weeks mating period in both sexes, approximately 3 weeks postmating in males, and the entire gestation period as well as 21 days of lactation and up to 15 days postweaning, or 38 days postmating for sperm negative females.
In addition groups of 10 males and 10 females, selected from F1 pups to become F1 rearing animals, were treated with the test substance at doses of 0, 100, 300 and 1000 mg/kg bw/d postweaning until puberty. Parameters examined are detailed under "Examinations".
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 010545EDA0
- Expiration date of the lot/batch: Jan 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator, avoid temperatures > 35 °C
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The test guidelines require the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 36 +/- 1 days
- Weight at study initiation: males: 118.4-142.6 g, females: 92.7-124.3 g
- Fasting period before study: no
- Housing: as groups of 4 in Polysulfonate cages supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany with exceptions: during overnight matings, male and female mating partners were housed together in Polycarbonate cages type III (same supplier) and pregnant animals and their litters were housed together until PND 21 in Polycarbonate cages type III. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ Lignocel® block large, J. Rettenmaier & Söhne
GmbH + Co KG, Rosenberg, Germany) were added. In addition in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherlands) were added.
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, drinking water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water (with 10 mg/ 100 mL Cremophor EL)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with drinking water with 10 mg/100 mL Cremophor EL and intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 1, 3 and 10 g/100 mL
- Amount of vehicle: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight until there is evidence of copulation or the maximum period of 14 days has elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: single and later together with pups
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in drinking water + 10 mg/100 mL Cremophor EL for a period of 7 days at room temperature were carried out prior to the start of the study.
At the beginning and at the end of premating, once during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control analysis. Of each sample, one additional reserve sample was retained.
The samples collected at the beginning of the administration period and during lactation period were analyzed. The remaining samples were stored at -20 °C.

Results:
The stability of test substance in drinking water + 10 mg/100 mL Cremophor EL was demonstrated for a period of 7 days at room temperature.
The homogeneous distribution of the test substance in drinking water + 10 mg/100 mL Cremophor EL was demonstrated.
Measured values for the test item were in the expected range of the target concentrations (90 - 110 %), demonstrating the correctness of the preparations. Measured values of samples 30 - 32 were slightly under the specification limit of 90 % (average 88.7 %), however, the re-check using the retain samples 30R-32R proved to be within the specification limits of 90-110 % (average 92.2 %).
Duration of treatment / exposure:
Animals of parental generation were treated for:
females: 126/133 days
males: 109/110 days
F1 rearing animals:
from PND 21 till sexual maturation: female: PND 21 to 36, male PND 21 to 47

The duration of treatment covered a 10 weeks premating period and 2 weeks mating period in both sexes, approximately 3 weeks postmating in males, and the entire gestation period as well as 21 days of lactation and up to 15 days postweaning, or 38 days postmating for sperm negative females.
F1 rearing animals were treated from postweaning until puberty.
Frequency of treatment:
once daily, 7 days per week, exception: no administration to animals being in labor
Details on study schedule:
- No pups were selected for mating as a OECD 422 study was conducted.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 parental animals
10 F1 rearing animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on request of the sponsor.
Positive control:
No positive control conducted.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the administration period all animals were checked daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in all animals once prior to the first administration (day 0) and at weekly intervals thereafter.
- Detailed clinical observations checked in table No.1 were included.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 10 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined once a week for male and female parental animals. Food consumption was not determined after the 10th premating week (male parental animals) and during the mating period (male and female parental animals). Food consumption of the females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14, and 14-20. Food consumption of females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes, as part of the detailed neurological examination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males: at termination, females: at PND 14
- Anaesthetic used for blood collection: Yes, isoflurane
- How many animals: in the first 10 surviving parental males (fasted) per group and a maximum of 10 females (fasted) with litters (in order of delivery) per group
- Parameters checked in table No.5 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males: at termination, females: at PND 14
- Animals fasted: Yes
- How many animals: in the first 10 surviving parental males per group and a maximum of 10 females with litters (in order of delivery) per group
- Parameters checked in table No.6 were examined.

URINALYSIS: Yes, as part of detailed clinical observations.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: end of administration period
- Dose groups that were examined: all, first 5 parental males and the first 5 females with litter (in order of delivery) per group
- Battery of functions tested are checked into table No. 2-4. In addition motor activity was determined in the dark using the TSE Labmaster System (TSE Systems GmbH, Bad Homburg, Germany) with 18 infrared beams per cage. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes.

IMMUNOLOGY: No

OTHER: Thyroid hormones
Blood samples for T4 and TSH were collected from retrobulbar venous plexus under isoflurane anesthesia.
- Time schedule for collection: from all dams at PND 14 and all males at termination, animals were fastened
- Dose groups that were examined: all males
Oestrous cyclicity (parental animals):
For a minimum of 3 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight (all males)
determined in the right testis or right epididymis: sperm motility (all males), sperm head count (caud epididymis and testis) and sperm morphology (control and highest dose group)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); The surplus pups or 2 preferably female pups per litter were sacrificed under isoflurane anesthesia by decapitation.
Standardization of litters was not performed in litters with <= 8 pups

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: yes
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: only as general observation

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

OTHER: Thyroid hormones
Blood samples were taken from all surplus pups pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia.
Blood samples from the PND 13 pups were assessed for serum levels for T4 and TSH.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed 110/111 days after the beginning of the administration.
- Maternal animals: All surviving animals were allowed to litter and rear their pups until PND 21. They were sacrificed 127/134 days after the beginning of the administration.

GROSS PATHOLOGY: Yes. All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights: Were determined as listed in table No. 7.

HISTOPATHOLOGY: Yes. The organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution as listed in table No. 8. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
Postmortem examinations (offspring):
SACRIFICE and GROSS NECROPSY and HISTOPATHOLOGY / ORGAN WEIGTHS
On PND 4 the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.

On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4 % formaldehyde solution and were further processed.

On PND 21, the surplus F1 generation pups that were not used as F1 rearing animals were likewise be sacrificed under isoflurane anesthesia with CO2. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.

All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
Statistics:
Means and standard deviations were calculated.
Further statistics were performed as listed in table No. 9.
Reproductive indices:
Following indices were determined:
Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index, Live birth index, Postimplantation loss
Offspring viability indices:
Following indices were determined:
Viability index, Lactation index, Sex ratio, Anogenital index
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All male and all female animals of the mid- and high-dose groups (300 and 1000 mg/kg bw/d) showed salivation at least on one occasion during the treatment period. Individual males (three) of the low-dose group occasionally showed salivation during the mating and postmating periods. Due to salivation several male and female animals of the high- and mid-dose groups showed red discolored fur in the mouth or nose region during the treatment.
The temporary salivation and discolored fur was considered to be test substance-induced. From the temporary, short appearance of salivation immediately after dosing it is likely, that these findings were 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.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups (01 - 03 , 100, 300 and 1000 mg/kg bw/d) during the study.
There were sporadic findings in individual F0 animals as follows:
One high-dose male animal (No. 43) showed protruding eyeball during mating days 4 - 14 and postmating days 0 - 22. One mid-dose male animal (No. 27) showed hyperthermia and sparse fur (grade: severe) during mating days 1 - 14 and during postmating days 0 - 2 and 0 - 21, respectively. One sperm positive high-dose female (No. 138) and one sperm negative middose female (No. 136) did not deliver F1 pups. None of these findings is considered to be treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test substance-related mortalities in any of the groups.
One F0 female of test group 03 (1000 mg/kg bw/d) showed piloerection and vaginal discharge (light yellow) on GD 19 and was found dead on GD 20. Histopathology revealed a marked inflammation of the placenta. This was considerd to be a spontaneous finding.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights and body weight change of all male and female F0 generation parental animals in all test substance-treated groups were comparable to the concurrent control during the entire study period.
The statistically significantly lower body weight change in the mid-dose males during premating days 63 – 70, the statistically significantly higher body weight change in the mid-dose females during premating days 38 - 35 as well as significantly higher body weight change in the mid and high-dose females during PND 0 - 21 were considered as spontaneous in nature and not as treatment related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of all test substance-treated F0 male and female animals (100, 300 and 1000 mg/kg bw/d) was not influenced by the treatment throughout the entire study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related findings were observed.
One high-dose male animal (No. 43) showed protruding eyeball during mating days 4 - 14 and postmating days 0 - 22. In the pupillary reflex test one out of 5 examined male animals of dose group 01 and two out of 5 examined male animals of dose group 03 showed a retarded adaption of the pupil to light. These findings were regarded as spontaneous findings.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period in males of test group 03 (1000 mg/kg bw/d) red blood cell (RBC) counts and hematocrit values were slightly, but significantly decreased.
Additionally, in males of test group 03 (1000 mg/kg bw/d) absolute eosinophil cell counts were slightly lower compared to controls, but this was the only changed differential blood cell fraction and therefore this alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). In females of test group 03 (1000 mg/kg bw/d) at lactation day 14, absolute and relative neutrophil counts were significantly lower and relative lymphocyte counts were significantly higher compared to controls. No changes among the total white blood cell (WBC) counts and other hematology parameters occurred in these individuals. Therefore, these alterations were regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).
In males of test groups 02 and 03 (300 and 1000 mg/kg bw/d) after the administration period, prothrombin time (Hepatoquick’s test, HQT) was significantly reduced, but the values were within the historical control range (males, HQT 34.6-40.2 sec). Therefore, this change was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
After the administration period in males of test group 03 (1000 mg/kg bw/d), total protein and globulin values as well as cholesterol and potassium values were significantly increased.
After lactation day 14 in females of test group 03 (1000 mg/kg bw/d) also total protein and globulin levels as well as albumin values were significantly higher compared to controls. Additionally, in females of test group 03 (1000 mg/kg bw/d) creatinine values were significantly lower compared to controls.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.
After the administration period in males of test groups 02 and 03 (300 and 1000 mg/kg bw/d), urine pH values were significantly decreased whereas urine specific gravity was significantly increased. Additionally, in males of test group 03 (1000 mg/kg bw/d) incidences of ketone bodies were significantly higher compared to controls. No hint of a dysregulation of the energy metabolism (e.g. changes in serum glucose, creatinine, triglycerides) was observed among these individuals. It can be assumed, that acidic metabolites of the compound are excreted via the urine decreasing the urine pH and maybe increasing the urine specific gravity. Methacrylic acid as metabolite of the compound is further degraded to Acetyl-CoA, and high abundance of this molecule results in formation of acetoacetate as ketone body, which is excreted via the urine (Greim et al., 1995). These changes are significant only in males, because metabolism rate is normally higher in this sex. Therefore, the mentioned altered urine parameters in males of test groups 02 and 03 are regarded as treatment-related, but not as adverse.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

The open field observations did not reveal any test substance-related findings in F0 male and female animals of test groups 01 and 02.
Three out of 5 examined male animals of dose group 03 (1000 mg/kg bw/d) showed slight salivation (area around the mouth was moist), which was considered to be treatment-related.

There were no test substance-related findings in male and female F0 animals of all test groups in sensorimotor tests/reflexes.
In the pupillary reflex test one out of 5 examined male animals of dose group 01 and two out of 5 examined male animals of dose group 03 showed a retarded adaption of the pupil to light. One out of 5 examined male animals of dose groups 02 and 03, respectively, showed very frequent vocalizations when touched. These were regarded as spontaneous findings.

No test substance-related impaired quantitative parameters were observed in male and female animals of all test groups.
As there was no dose-response the statistically significantly lower values of grip strength of forelimbs in females of dose group 01 was considered as spontaneous in nature and not treatment-related.

No statistically significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in in the liver in female animals of test group 03.
In female animals, minimal hepatocellular hypertrophy was noted in the liver, with incidences and grading according to the table 12.
In male animals, minimal follicular hypertrophy/hyperplasia was noted in the thyroid gland, with incidences and grading according to the table 13.

In females, the predominant pattern of hepatocellular hypertrophy was centrilobular, the animal showing the periportal pattern was the premature decedent (female animal 137), where the liver was markedly enlarged even on gross evaluation. This was the only animal examined during pregnancy, therefore this possibly confounding factor should be taken into account and the relationship to treatment of the observed periportal hypertrophy is questionable.

The findings observed in the thyroid gland in male animals of test group 03 were finally considered as secondary in nature due to the observed functional liver effects and thus, as not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
One male animal of test group 03 (animal No. 41) showed a lymphoma, this was assessed as incidental, as the occurrence of lymphoma has been documented in single control animals of this age in this laboratory.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones:
In parental males (test groups 01, 02 and 03; 100, 300 and 1000 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data revealed regular cycles in the parental females of all test groups including the control. The mean estrous cycle duration in the different test groups (00 - 03) was between 4.0 and 4.1 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
For motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no
treatment-related effects were observed in any of the test groups.
The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls.
Reproductive performance:
no effects observed
Description (incidence and severity):
- Male reproduction data
For nearly all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Copulation was not confirmed for mid-dose male No. 36 paired with mid-dose female No. 136.
Thus, the male mating index was 91.7 % in the mid-dose group and 100 % in the control, low- and high-dose groups.

Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One high-dose male (No. 38) did not generate pregnancy.
Thus, the male fertility index ranged between 91.7 % and 100 % without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

- Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 91.7 % in test group 02 and 100 % in test groups 00 - 01 and 03.

The mean duration until sperm was detected (GD 0) varied between 2.2 and 3.8 days without any relation to dosing.

All female rats delivered pups or had implants in utero (exceptions: 300 mg/kg bw/d female No. 136 (mated with male No. 36) did not become pregnant; 1000 mg/kg bw/d female No. 138 (mated with male No. 38) did not become pregnant).
The fertility index varied between 100 % in test groups 00, 01 and 02 and 91.7 % in test group 03. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.

The mean duration of gestation was similar in all test groups (i.e. between 21.9 and 22.0 days). The gestation index was 100 % in test groups 00 and 02, 91.7 % in test group 01 and 90.9 in test group 03, indicating no treatment-related changes.

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (11.2 / 11.9 / 12.7 and 11.4 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (10.5 / 11.3 / 12.0 and 10.6 pups/dam in test groups 00 - 03, respectively).

The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100 % / 100 % / 100 % and 98.1 % in test groups 00 - 03. Moreover, the number of stillborn pups was not significantly different between the test groups.

In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
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 examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- F1 pups
There were no test substance-related, adverse clinical signs observed in any of the F1 generation pups of the different test groups.
One male pup (No. 2) of high-dose dam No. 142 had an injury (red mouth region) and showed lateral position on PND 7. This is considered to be a spontaneous event.

- F1 rearing animals
All male and all female F1 rearing animals of the high- and all male and several female F1 rearing animals of the mid-dose groups (300 and 1000 mg/kg bw/d) showed salivation at least on one occasion during the treatment period. Due to salivation several male and female animals of the high- and mid-dose groups showed red discolored fur in the mouth or nose region during the treatment.
The temporary salivation and discolored fur was considered to be test substance-induced. From the temporary, short appearance of salivation immediately after dosing it is likely, that these findings were 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.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F1 rearing animals in any of the groups (01 - 03 , 100, 300 and 1000 mg/kg bw/d) during the study.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- F1 pups
In the high-dose group (1000 mg/kg bw/d) 11 pups were found dead or cannibalized shortly after birth (PND 1) vs 1 in the control group. However, 10 of these decedents were from one single, rather large litter (No. 146, altogether 14 pups). There were no other findings regarding pup mortality in this dose group. Thus, the viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 99.4 % / 98.7 % / 100 % and 91.2 % in test groups 00 - 03, respectively.
The survival index indicating pup mortality during lactation (PND 4 - 13) varied between 100 % / 100 % / 100 % and 98.8 % in test groups 00 - 03, respectively.

- F1 rearing animals
There were no test substance-related mortalities in any of the groups. One selected F1 male rearing pup of test group 02 died on PND 22 due to a gavage error. This pup was replaced (dam No. 130, male pup No. 4).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- F1 pups
Mean body weights of the high-dose (1000 mg/kg bw/d) F1 male and female pups combined were statistically significantly below the concurrent control on PND 7 (about 11 % below control) and on PND 13 (about 9 % below control) but partly recovered until weaning (PND 21, about 6 % below control, non-significant).
High-dose pups gained only transiently less weight than the concurrent control between PND 4 - 7 (about 18 % less) with an intermittent effect on pup weights. However, pup weight and weight gain recovered under treatment and were close to the control level again during remaining lactation until weaning.
No effects on pup body weight/pup body weight gain were seen in the low- and mid-dose groups.
One male runt was seen in test group 01, one female runt was seen in test group 02 and three male and two female runts were seen in test group 03. These runts had no influence on the average weight of the newborn in any of the test groups.

- F1 rearing animals
The body weights and body weight change of all test substance treated male and female F1 rats (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control values throughout the entire study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- F1 rearing animals
Food consumption of all test substance treated male and female F1 rats (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control values throughout the entire study.
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
Description (incidence and severity):
- F1 rearing animals
Vaginal opening
Each female F1 rearing animal was evaluated for commencement of puberty. The first day when vaginal opening was observed was PND 29, the last was PND 36. The mean number of days to reach the criterion in the control and 100, 300 and 1000 mg/kg bw/d test groups amounted to 31.7, 31.6, 32.0 and 32.0 days. The mean body weight on the day, when vaginal opening was recorded, amounted to 100.1 g, 102.2 g, 102.0 and 97.7 g in test groups 0, 100, 300 and 1000 mg/kg bw/d. None of these values indicated any treatment-related effect.

Preputial separation
Each male F1 rearing animal was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 47. The mean number of days to reach the criterion in the control and 100, 300 and 1000 mg/kg bw/d test groups amounted to 41.7, 42.3, 41.3 and 41.7 days. The mean body weight on the day, when preputial separation was recorded, amounted to 180.5 g, 176.1 g, 177.8 and 166.7 g in test groups 0, 100, 300 and 1000 mg/kg bw/d. None of these values indicated any treatment-related effect.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- F1 pups
A few F1 pups showed spontaneous findings at gross necropsy, such as absent testis (left), thorax fluid-filled (red), dilated renal pelvis, post mortem autolysis, absent epididymis (left), incisors sloped (upper and lower), empty stomach and extended intestine. These findings occurred without any relation to the dose and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

- F1 rearing animals
One male mid-dose F1 rearing animal showed the spontaneous finding small testis (both) at gross necropsy. This finding occurred without any relation to dosing and can be found in the historical control data at comparable or even higher incidences. Thus, this finding was not considered to be associated to the test substance.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- F1 pups
Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Anogenital distance/anogenital index
Neither anogenital distance nor anogenital index were affected in any of the test substance treated F1 male and female pups (test groups 01 - 03 [100, 300 and 1000 mg/kg bw/d]).

Nipple/ areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13. However, all animals were rechecked for nipples/areolae on PND 20, one day prior to weaning. During this re-examination no areolae were detected in any male pup in all test groups (01 - 03).

Thyroid hormones
In male and female pups at PND13 (100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
no

Table No. 10 Absolute organ weights

When compared with control group 00 (=100%), the following mean absolute weights were significantly increased in test group 03:

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Terminal body weight

101 %

96 %

93 %

101 %

102 %

100 %

Liver

99 %

101 %

123 %**

101 %

108 %

122 %**

Kidney

94 %

94 %

113 %**

104 %

105 %

117 %**

*p ≤ 0.05; **p ≤ 0.01

 

Table No. 11 Relative organ weight

When compared with control group 00 (=100%), the following mean relative organ weights were significantly increased or decreased in one or more test groups:

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Adrenal glands

103 %

106 %

123 %**

-

-

-

Kidneys

93 %

97 %

120 %**

102 %

102 %

117 %**

Liver

98 %

105 %**

131 %**

100 %

105 %

122 %**

Pituitary gland

102 %

105 %

114 %**

-

-

-

Spleen

86 %*

91 %

127 %

-

-

-

*p ≤ 0.05; **p ≤ 0.01

 

Table No. 12 Histopathology

Liver

Female animals

Test group

(mg/kg bw/d)

0 (control)

1 (100)

2 (300)

3 (1000)

No. of animals

12

12

12

12

Hypertrophy, centrilobular

0

0

0

3

Grade 1

 

 

 

3

Hypertrophy, peripheral

0

0

0

1

Grade 2

 

 

 

1

 

Table No. 13 Histopathology

Thyroid gland

Male animals

Test group

(mg/kg bw/d)

0 (control)

1 (100)

2 (300)

3 (1000)

No. of animals

12

12

12

12

Hypertrophy/hyperplasia, follicular

0

0

0

2

Grade 1

 

 

 

2

DISCUSSION

In this Modified Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (OECD 422), the test substance cyclohexyl methacrylate was administered daily as an aqueous preparation to groups of 12 male and 12 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) to screen for potential repeated dose, reproductive and developmental toxicity. The duration of treatment covered a 10 weeks premating period and 2 weeks mating period in both sexes, approximately 3 weeks postmating in males, and the entire gestation period as well as 21 days of lactation and up to 15 days postweaning, or 38 days postmating for sperm negative females.In addition groups of 10 males and 10 females, selected from F1 pups to become F1 rearing animals, were treated with the test substance at doses of 0, 100, 300 and 1000 mg/kg bw/d postweaning until puberty. The study was terminated with the terminal sacrifice of the rearing animals. Thus, the reliability regarding the possible reproductive and developmental properties was increased due to the longer premating treatment period (10 weeks) and a postweaning follow-up of selected offspring until puberty.

Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was demonstrated over a period of 7 days under ambient conditions.

The majority of male and female F0 and F1 animals in test groups 3 and 2 (1000 mg/kg bw/d and 300 mg/kg bw/d) showed salivation for a time period after gavage treatment. Secondary to salivation several male and female animals of these test groups showed red discolored fur in the mouth or nose region during the treatment. From the temporary, short appearance of salivation 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. As such, this local finding was not considered to be an adverse and systemic-toxicologically relevant effect. While occasional salivation was the only notable finding during detailed clinical observations, no abnormalities were noted in FOB or motor activity measurements.

Food consumption and body weights/body weight gain remained unaffected in all treatment groups, both in F0 parents and postweaning in F1 rearing animals.

Dysregulation of the liver cell metabolism was detected in males and females treated with 1000 mg/kg bw/d, which was seen as increase of serum total protein and globulin values (males and females), increase of cholesterol and potassium levels (males) and high albumin level and low creatinine values (females). Additionally a marginal anemia was observed because of decreased red blood cell (RBC) counts and hematocrit values. Furthermore the liver showed a marked weight increase in animals of both sexes treated with 1000 mg/kg bw/d. A corresponding histopathological correlate in the form of hepatocellular centrilobular hypertrophy could only be detected in female rats. No further adverse or primary substance-related effects were detected in animals at 1000 mg/kg bw/d. At 100 and 300 mg/kg bw/d no test substance-related adverse findings were determined.

Under the conditions of this study the test compound had no adverse effects on fertility and reproductive performance of the F0 parental animals of both sexes up to 1000 mg/kg bw/d as mating behavior, conception, implantation, delivery and rearing of offspring were not influenced.

High-dose pups gained only transiently less weight than the concurrent control between PND 4 - 7 (about 18 % less) with an intermittent effect on pup weights. However, pup weight and weight gain recovered under treatment and were close to the control level again during remaining lactation until weaning. In the selected F1 rearing animals, there were neither effects on food consumption or body weight, nor on the timing of puberty in the F1 rearing animals. Altogether, neither pre- and postnatal developmental toxicity nor any effects on anogenital distance/index, presence of nipples or sexual maturation were noted in all treatment groups.

 

CONCLUSION

Under the conditions of the present modified combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats (OECD 422/408), the NOAEL for general, systemic toxicity of Cyclohexyl methacrylate was 300 mg/kg bw/d for male and female rats, based on functional impairment in rats at 1000 mg/kg. The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/d for the F0 parental rats and the NOAEL for developmental toxicity in the F1 progeny was 1000 mg/kg bw/d.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was conducted according to guideline and GLP.
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
610 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The weight of evidence is considered appropriate and the quality of the data is sufficient for assessment.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

One oral Modified Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (OECD 422/408) using the test substance cyclohexyl methacrylate is available. Furthermore a study with the lower methacrylate ester methyl methacrylate is used to support the assessment of reproduction toxicity of cyclohexyl methacrylate.

 

For the inhalation route no studies on reproduction toxicity of cyclohexyl methacrylate are available, therefore studies of the hydrolysis products cyclohexanol, methacrylic acid and the lower methacrylate ester methyl methacrylate were used to assess the potential of cyclohexyl methacrylate for reproduction toxicity via inhalation and support the observations from the oral route.

 

Key study

Oral exposure

A “Modified Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” in rats was conducted according to OECD 422 and OECD 408 using the test substance. Cyclohexyl methacrylate was administered daily as an aqueous preparation to groups of 12 male and 12 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day. Control animals were dosed daily with the vehicle only (drinking water + 10 mg/100 mL Cremophor EL). The duration of treatment covered a 10 weeks premating period and 2 weeks mating period in both sexes, approximately 3 weeks postmating in males, and the entire gestation period as well as 21 days of lactation and up to 15 days postweaning, or 38 days postmating for sperm negative females.

Groups of 10 males and 10 females, selected from F1 pups to become F1 rearing animals, were treated with the test substance at doses of 100, 300 and 1000 mg/kg bw/d postweaning until puberty. The study was terminated with the terminal sacrifice of the rearing animals. Thus, the reliability regarding the possible reproductive and developmental properties was increased due to the longer premating treatment period (10 weeks) and a postweaning follow-up of selected offspring until puberty.

After 10 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females.

A detailed clinical observation (DCO) was performed in all animals before the start of the administration period and thereafter at weekly intervals. Food consumption of the F0 parents and F1 rearing animals was determined once weekly during premating. In dams, food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10, 10 - 13, 13 - 17 and 17 - 21. Body weights of F0 parents and F1 rearing animals were determined once a week. Estrous cycle data were evaluated for F0 generation females over a three-week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7, 13 and 21. Their viability was recorded. At necropsy on PND 4, 13 and 21, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. Anogenital distance measurements were conducted in a blind randomized fashion on all live male and female pups on PND 1. All surviving male pups were examined for the presence or absence of nipple/areola anlagen on PND 13 and were re-examined on PND 20. The number of nipple/areola anlagen were counted. Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement.

Clinico-chemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group.

All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 rearing animals was recorded. All F1 rearing animals were assessed macroscopically.

 

The stability, the homogeneous distribution and correct concentrations of the test substance in drinking water + 10 mg/100 mL Cremophor EL were demonstrated by analysis.

The majority of male and female F0 and F1 animals in test groups 3 and 2 (1000 mg/kg bw/d and 300 mg/kg bw/d) showed salivation for a time period after gavage treatment. Secondary to salivation several male and female animals of these test groups showed red discolored fur in the mouth or nose region during the treatment. From the temporary, short appearance of salivation 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. As such, this local finding was not considered to be an adverse and systemic-toxicologically relevant effect. While occasional salivation was the only notable finding during detailed clinical observations, no abnormalities were noted in FOB or motor activity measurements.

Food consumption and body weights/body weight gain remained unaffected in all treatment groups, both in F0 parents and postweaning in F1 rearing animals.

Dysregulation of the liver cell metabolism was detected in males and females treated with 1000 mg/kg bw/d, which was seen as increase of serum total protein and globulin values (males and females), increase of cholesterol and potassium levels (males) and high albumin level and low creatinine values (females). Additionally, in F0 males of test group 03 (1000 mg/kg bw/d) a marginal anemia was observed. Furthermore the liver showed a marked weight increase in animals of both sexes treated with 1000 mg/kg bw/d. A corresponding histopathological correlate in the form of hepatocellular centrilobular hypertrophy could only be detected in female rats. No further adverse or primary substance-related effects were detected in animals at 1000 mg/kg bw/d. At 100 and 300 mg/kg bw/d no test substance-related adverse findings were determined.

Under the conditions of this study the test compound had no adverse effects on fertility and reproductive performance of the F0 parental animals of both sexes up to 1000 mg/kg bw/d as mating behavior, conception, implantation, delivery and rearing of offspring were not influenced.

High-dose pups gained only transiently less weight than the concurrent control between PND 4 - 7 (about 18 % less) with an intermittent effect on pup weights. However, pup weight and weight gain recovered under treatment and were close to the control level again during remaining lactation until weaning. In the selected F1 rearing animals, there were neither effects on food consumption or body weight, nor on the timing of puberty in the F1 rearing animals. Altogether, neither pre- and postnatal developmental toxicity nor any effects on anogenital distance/index, presence of nipples or sexual maturation were noted in all treatment groups.

Under the conditions of the present modified combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats (OECD 422/408), the NOAEL (no observed adverse effect level) for general, systemic toxicity of Cyclohexyl methacrylate was 300 mg/kg bw/d for male and female rats, based on functional liver impairment in F0 parental rats at 1000 mg/kg.

The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/d for the F0 parental rats.

The NOAEL for developmental toxicity in the F1 progeny was 1000 mg/kg bw/d.

 

Supporting studies

Oral exposure

Methyl methacrylate

Methyl methacrylate (CAS 80-62-6) has been tested in a reliable two-generation reproduction toxicity study in rats with oral administration (gavage). The study was performed according to OECD TG 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) did not show 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.

 

Inhalation exposure

Cyclohexanol

To address potential reproductive concerns of cyclohexanol, a modified OECD 422 guideline study was performed (US EPA, 2010). A separation into a repeated dose and a reproductive component was done.In the repeated dose component 15 animals/sex/dose were whole body exposed to cyclohexanol vapours at concentrations of 0, 50, 150 or 450/400 ppm (0; 0.21; 0.61; 1.84/1.64 mg/L). For males the exposure time was 6 hours/day, 5 days/week over a period of 16 weeks (10 weeks premating to completely cover the spermatogenesis). 5 animals/sex/dose were allowed to recover for a period of 4 weeks. The females were exposed 6 hours/day, 5 days/week for the first 10 weeks and 6 hours/day, 7 days/week for the next 6 weeks. Further, the modifications to the original OECD 422 protocol included the investigation of sperm parameters.

No test article-related effect was evident during the premating, gestation, or lactation periods on body weight, body weight gain, reproductive and fertility parameters, and litter size data. Lower testicular homogenization-resistant sperm head counts, Daily Sperm Production (DSP) and DSP/gram tissue values in the terminal 450/400 ppm animals were not considered toxicologically meaningful since values were within the range of recent historical control data. Pup sex ratios and pup survival to Lactation Day 4 were also unaffected by treatment. Two of the 11 pregnancies (18.2 %) in the 450/400 ppm group resulted in no viable pups at parturition. Lower mean pup body weights were also seen at birth and Postnatal Day 4 in this high exposure group. The NOAEC for reproductive toxicity was set to 0.610 mg/L.

 

Conclusions

Based on the reproduction toxicity study in rats with cyclohexyl methacrylate the no observed adverse effect level (NOAEL) for fertility and reproductive performance was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 progeny was 1000 mg/kg bw/d

 

The result of the oral study conducted with the test substance is also supported by the available oral study conducted with Methyl methacrylate. This study underlines the absence of adverse effects for the fertility or reproductive performance of the P or F1 parental animals up to and including a dose of 400 mg/kg bw/day. This value is further supported by oral reproductive screening studies of other lower methacrylate esters (please refer to read across justification) resulting in similar NOAEL values for fertility of rats.

For inhalation exposure with the hydrolysis product cyclohexanol the only treatment-related effects seen occurred at the highest, initially lethal, test level and included a slight increased incidence of pregnancies with no viable pups at birth and lower F1 pup weights on the highest dosing group.

Effects on developmental toxicity

Description of key information

oral

Cyclohexyl methacrylate, rabbit: NOAEL maternal toxicity 500 mg/kg bw/d; NOAEL developmenatal toxicity 500 mg/kg bw/d (BASF SE 2018)

Cyclohexyl methacrylate, OECD 408/422, rat NOAEL developmenatal toxicity 1000 mg/kg bw/d (BASF SE 2018)

analogous substance methyl methacrylate (CAS# 80-62-6), rabbit: NOAEL maternal toxicity 50 mg/kg bw; NOAEL fetotoxicity ≥ 450 mg/kg bw ; NOAEL teratogenicity > 450 mg/kg bw  (BASF SE, 2009) inhalation

analogous substance methyl methacrylate (CAS# 80-62-6), rat: LOEC maternal toxicity ca. 0.41 mg/L (corresponding to 99 ppm) ; due to body weight reduction); NOAEC fetotoxicity, teratogenicity ≥ ca. 8.3 mg/L (corresponding to ca. 2028 ppm; Rohm & Haas, 1991)

analogous substance methyl methacrylate (CAS# 80-62-6), mice: no maternal toxicity was observed (no NOAEC given); NOAEC fetotoxicity, teratogenicity ≥ 1.64 mg/L; no embryotoxic or teratogenic effects observed (Rohm & Haas, 1976)

hydrolysis product cyclohexanol (CAS#  108-93-0), rat: NOAEC maternal toxicity 610 mg/m³; based on mortality in high dose group; NOAEC fetotoxicity, teratogenicity 610 mg/m³; based on a increased incidence of pregnancies with no viable pups at birth and lower F1 pup body weights (US EPA, 2010)

hydrolysis product methacrylic acid (CAS# 79-41-4), rat: NOAEC maternal toxicity 716 mg/m³; based on decreased body weight and food consumption; NOAEC fetotoxicity, teratogenicity 1076 mg/m³; no embryotoxic or teratogenic effects oberved (Saillenfait et al., 1999)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-14 to 2017-09-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008, Part B: Methods for the determination of toxicity and other health effects: Prenatal Developmental Toxicity Study; Official Journal of the European Union, No. L 142
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 010545EDA0
- Expiration date of the lot/batch: Jan 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator, avoid temperatures > 35 °C
Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl:KBL(NZW)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France
- Age at study initiation: 29-38 days
- Weight at study initiation: 3046 – 4305 g
- Fasting period before study: no
- Housing: single, Cage System, Type 4X03B700CP, with Wooden gnawing blocks (Typ KNH E-041), Abedd®, Lab. and Vet. Service GmbH, Vienna, Austria
- Diet: ad libitum, Pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, Provimi Kliba SA, Kaiseraugst/Switzerland
- Water: ad libitum, tap water
- Acclimation period: at least 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water (with 10 mg/ 100 mL Cremophor EL)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the test substance preparations, the specific amount of test substance was weighed, topped up with drinking water (with 10 mg/ 100 mL Cremophor EL) in an Erlenmeyer flask and intensely mixed with a homogenizer.

- Rate of preparation:The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability.
- Storage temperature: Room temperature

VEHICLE
- Justification for use and choice of vehicle: ??Cremophor EL was added to enhance water solubility. ??
- Concentration in vehicle: 0.5, 1.5, 5.0 g/100 mL
- Amount of vehicle: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water (10 mg/100 mL Cremophor EL) over a period of 7 days at room temperature had been verified prior to the start of the study in a similar batch.
Samples of the test substance preparations were sent to the analytical laboratory once during the study period (at the beginning of administration) for verification of the concentrations. HPLC was used for the analysis. The samples taken for the concentration control analyses were also used to verify the homogeneity of the samples of the low- and high-concentrations each (50 and 500 mg/kg bw/d). Three samples (one from the top, middle and bottom) were taken for each of these preparations from the preparation vessel with a magnetic stirrer running.
The mean measured concentrations of the samples (mean values of the samples 3-5 and 7-9 as well as the single value of the sample 6) of treatment groups corresponded to the expected values within the limits of the analytical method, i.e. were above 90 % and below 110 % of the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: artificial insemination
A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal) was injected intramuscularly to the female rabbits about 1 hour before insemination.
The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females.
Each female was inseminated with the sperm of a defined male donor as documented in the raw data. 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 GD 0 (beginning of the study) and the following day as GD 1.
Duration of treatment / exposure:
23 days (GD 6 - 28)
Frequency of treatment:
once daily
Duration of test:
30 days (GD 0 - 29)
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females per group, no males
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The high dose was selected based on signs of toxicity noted at dose levels of 300 and 1000 mg/kg bw/d in a previously conducted maternal toxicity range-finding study which preceded this definitive prenatal developmental toxicity study.
In the maternal toxicity range‐finding study, 5 pregnant New Zealand White rabbits per dose were administered the test substance by oral gavage from gestational day (GD) 6 through GD 28.
At 1000 mg/kg bw/d food consumption was reduced by an average of 40 % during treatment, in mid-gestation food consumption was actually less than half the normal intake. At 300 mg/kg bw/d food consumption was still decreased by about 20 %. Body weights were lower as well in both treatment groups, final weights were 10 % and 7 % below control, respectively. Corrected (net) body weight change was 4 times lower at 1000 mg/kg bw/d than in controls. Necropsy revealed abnormal content (whitish plastic-like streaks intermingled in food) in the stomach in 4 of 5 high-dose does. From the appearance of the stomach content it seems likely that this high concentration of test substance polymerizes under acidic conditions in the stomach and in/or the presence of food ingredients. Based on these data dose levels of 50, 150, and 500 mg/kg bw/d were considered to be appropriate for the present definitive study. The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily, during the administration period (GD 6 - 28) before the administration as well as within 5 hours after the administration
- Cage side observations: morbidity, pertinent behavioral changes and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29
- 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).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, recorded daily

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: uteri and ovaries
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Site of implantations
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

In the present study the internationally harmonized glossary of WISE et al. (1997) and the updated version MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI.
Statistics:
- Simultaneous comparison of all dose groups with the control group using the DUNNETTtest (two-sided) for the hypothesis of equal means:
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

- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions:
Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians:
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female of the low-dose group (50 mg/kg bw/d) and two females of the mid-dose group (150 mg/kg bw/d) had blood in bedding before and/or after treatment: doe No. 29 on GD 18, doe No. 54 on GD 20 and doe No. 67 on GD 18 (this animal died on the following day).

In total, reduced defecation was observed in eleven control, seven low-dose, nine mid-dose and thirteen high-dose females (0, 50, 150 and 500 mg/kg bw/d). No defecation was observed in six control females, five low-dose females, four mid-dose females and ten high-dose females.

Incidence and distribution of these findings do not indicate a relationship to the test substance.

There were no further clinical findings in the other does in this study.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
150 mg/kg bw/d : One female (No. 67) was found dead during the administration period (GD 19), after showing blood in bedding and reduced defecation on the days before. The gross pathological examination revealed multiple erosions in the stomach and no feces in rectum.

500 mg/kg bw/d: One female (No. 92) was sacrificed after abortion ahead of schedule (GD 27). Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, this was considered to be a spontaneous incident.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and the average body weight gain of the low-, mid- and high-dose groups (50, 150 and 500 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

Mean carcass weights and corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were not significantly different between all test groups including controls (0, 50, 150 or 500 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In comparison to the control group the mean food consumption of the does in test group 3 (500 mg/kg bw/d) was statistically significantly reduced at the beginning of the treatment period (GD 6-7), but recovered afterwards and was comparable again towards the end of the treatment period. During the treatment period (GD 6-28) the high-dose does consumed 6 % less food than the concurrent control does.

The food consumption of the low- and mid-dose rabbits (50 and 150 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.
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 examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean gravid uterus weight of the rabbits of test groups 1-3 (50, 150 or 500 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some spontaneous findings were noted in individual females of test groups 0-3 (0, 50, 150 and 500 mg/kg bw/d). These gross findings were:
- Single erosion in stomach in two control (Nos. 10 and 15), one low-dose (No. 35 - 50 mg/kg bw/d), one mid-dose (No. 61 - 150 mg/kg bw/d) and one high-dose doe (No. 100 - 500 mg/kg bw/d).
- Abnormal content in stomach in one high-dose doe (No. 86 - 500 mg/kg bw/d).
- Malpositioned kidney in one high-dose doe (No. 91 - 500 mg/kg bw/d).
- Absence of uterine horn(s) in one mid-dose doe (No. 69, not pregnant - 150 mg/kg bw/d).

Additional findings were noted in test groups 2 and 3. Both were associated with unscheduled maternal death or sacrifice:
- Multiple erosions in stomach and no feces in rectum in mid-dose doe No. 67 (died on GD 19).
- A stomach filled with very dry, hard feed, no feces in small intestine and rectum and a large intestine filled with fluid in high-dose doe No. 92 (sacrificed after abortion on GD 27).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One high-dose female (No. 92 - 500 mg/kg bw/d) was sacrificed after abortion ahead of schedule (GD 27). Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study this was considered to be a spontaneous incident.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the number of resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the number of resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the number of viable fetuses.
Changes in pregnancy duration:
not examined
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 examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate.
The conception rate was 92 % in the mid-dose group (150 mg/kg bw/d), 96% in the control and low-dose groups (0 and 50 mg/kg bw/d) and 100% in the high-dose group (500 mg/kg bw/d).
A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No test substance related systemic effects were observed.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the number of viable fetuses.
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 500 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The distribution of external malformations about the dose groups does not indicate an association to the treatment, no statistically significant differences between the groups were noted (table 1).
0 mg/kg bw/d: 1 fetus with open eye, 1 fetus with umbilical hernia
50 mg/kg bw/d: no findings
150 mg/kg bw/d: 2 fetuses with umbilical hernia
500 mg/kg bw/d: 1 fetus with malrotated limb, 1 fetus with cleft palate
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were detected in single fetuses of the test groups 1, 2 and 3 (50, 150, 500 mg/kg bw/d). Male high-dose fetus No. 82-09 had associated external and soft tissue malformations. The addition of those individual findings resulted in a slightly increased affected fetuses per litter-incidence of skeletal malformations in test group 3 (500 mg/kg bw/d), which became statistically significant as the control incidence was zero (table 7). However, as these were individual findings without any ontogenetic coherence and the high-dose incidence is even below the historical control mean (HCD: 1.4 mean% [0.0 - 4.4]), these findings were considered to be spontaneous in origin and not treatment-related.
0 mg/kg bw/d: no findings
50 mg/kg bw/d: 1 fetuses with fused skull bone
150 mg/kg bw/d: 1 fetuses with additional bony structure
500 mg/kg bw/d: 1 fetus with branched rib, 1 fetus with small palatine bone, 1 fetus with thoracic hemivertebra, misshapen thoracic vertebra, branched rib
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue malformations occurred in single fetuses of the test groups 2 and 3 (150 or 500 mg/kg bw/d). Male high-dose fetus No. 82-09 had additional external and skeletal malformations. No statistically significant differences between the groups were noted (table 4).
0 mg/kg bw/d: no findings
50 mg/kg bw/d: no findings
150 mg/kg bw/d: 1 fetuses with persistent truncus arteriosus, heart: muscular ventricular septum defect
500 mg/kg bw/d: 1 fetus with malpositioned lens, 1 fetus with small cerebrum
Other effects:
no effects observed
Description (incidence and severity):
- External:
One external variation was recorded in two fetuses of the same high-dose litter (500 mg/kg bw/d), i.e. paw hyperflexion (table 2). This finding can be found in the historical control data at comparable incidences, thus it is considered to be incidental.

Three unclassified external observations, i.e. amniotic fluid discolored, placentae fused and placentae necrobiotic, were recorded in one fetus, each, of test groups 0, 1 and 2 (0, 50 or 150 mg/kg bw/d) (table 3). These findings are not considered to be related to treatment.

- Soft tissue:
The examinations of the soft tissues revealed cystic dilatation of the brain, dilated cerebral ventricle and malpositioned carotid branches in individual fetuses of test groups 0, 1 and 3 (0, 50 or 500 mg/kg bw/d) (table 5). An absent lung lobe (Lobus inferior medialis) was noted in all substance-treated groups. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered
biologically relevant. All of them can be found in the historical control data of the test facility at comparable incidences.

One unclassified soft tissue observation was recorded in two control fetuses, six low-dose, four mid-dose and three high-dose fetuses: a blood coagulum around urinary bladder (table 6). This finding is not considered to be treatment-related.

- Skeletal:
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing (table 8 + 9). The overall incidences of skeletal variations were comparable to the historical control data.

Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (tabel 10). The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they were assessed as not treatment-related.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant adverse effects of the test substance were observed.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Total external malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

2 (0.9)

0

2 (0.9)

2 (1.0)

Litter incidence

N (%)

2 (8.3)

0

2 (9.1)

1 (4.2)

Affected fetuses/litter

Mean (%)

0.8

0

1.0

1.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 2: Total external variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

0

0

0

2 (1.0)

Litter incidence

N (%)

0

0

0

1 (4.2)

Affected fetuses/litter

Mean (%)

0

0

0

1.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 3: Total external unclassified observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

1 (0.4)

1 (0.4)

1 (0.5)

0

Litter incidence

N (%)

1 (4.2)

1 (4.2)

1 (4.5)

0

Affected fetuses/litter

Mean (%)

0.4

0.5

0.5

0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 4: Total soft tissue malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

0

0

1 (0.5)

2 (1.0)

Litter incidence

N (%)

0

0

1 (4.5)

2 (8.3)

Affected fetuses/litter

Mean (%)

0

0

0.6

0.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 5: Total soft tissue variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

3 (1.3)

3 (1.3)

5 (2.4)

5 (2.5)

Litter incidence

N (%)

3 (13)

3 (13)

3 (14)

4 (17)

Affected fetuses/litter

Mean (%)

1.4

1.6

2.6

2.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 6: Total soft tissue unclassified observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

2 (0.9)

6 (2.6)

4 (1.9)

3 (1.5)

Litter incidence

N (%)

2 (8.3)

4 (17)

3 (14)

2 (8.3)

Affected fetuses/litter

Mean (%)

1.9

3.2

2.2

1.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 7: Total skeletal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

0

1 (0.4)

1 (0.5)

3 (1.5)

Litter incidence

N (%)

0

1 (4.2)

1 (4.5)

3 (13)

Affected fetuses/litter

Mean (%)

0

0.3

0.6

1.2*

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent * = p0.05 (Wilcoxon-test [one-sided])

 

Table 8: Total fetal skeletal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

207 (91)

200 (86)

201 (95)

183 (91)

Litter incidence

N (%)

24 (100)

24 (100)

22 (100)

24 (100)

Affected fetuses/litter

Mean (%)

91.9

87.1

94.5

92.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 9: Occurrence of statistically significantly increased fetal skeletal variations

(expressed as mean percentage of affected fetuses/litter)

Finding

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

HCD

Mean % (range)

Incomplete ossification of hyoid; cartilage present

17.8

19.1

28.0*

16.0

31.1

(11.3-47.3)

Unossified talus; cartilage present

0

1.8*

1.7

0.7

1.0

(0.0-2.6)

Incomplete ossification of pubis; cartilage present

0

0

0.5

1.6*

0.7

(0.0-1.9)

 

Table 10: Total unclassified cartilage observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

18 (7.9)

14 (6.0)

21 (9.9)

15 (7.5)

Litter incidence

N (%)

8 (33)

7 (29)

7 (32)

8 (33)

Affected fetuses/litter

Mean (%)

6.8

5.3

10.2

6.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 11: Total fetal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

2 (0.9)

1 (0.4)

4 (1.9)

5 (2.5)

Litter incidence

N (%)

2 (8.3)

1 (4.2)

4 (18)

4 (17)

Affected fetuses/litter

Mean (%)

0.8

0.3

2.3

2.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 12: Total fetal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

207 (91)

200 (86)

201 (95)

184 (92)

Litter incidence

N (%)

24 (100)

24 (100)

22 (100)

24 (100)

Affected fetuses/litter

Mean (%)

91.9

87.1

84.5

92.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 13: Mean maternal body weights during gestation – grams

 

 

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Day 0

Mean

S.D.

N

3710 D

280.4

24

3739

256.3

24

3701

304.7

23

3736

248.0

25

Day 2

Mean

S.D.

N

3793 D

273.4

24

3815

260.5

24

3776

322.0

23

3801

253.7

25

Day 4

Mean

S.D.

N

3851 D

280.5

24

3875

269.3

24

3830

331.3

23

3847

257.3

25

Day 6

Mean

S.D.

N

3882 D

296.1

24

3916

279.5

24

3876

336.9

23

3885

285.4

25

Day 9

Mean

S.D.

N

3964 D

284.8

24

3993

264.7

24

3944

349.2

23

3942

286.3

25

Day 11

Mean

S.D.

N

4009 D

294.4

24

4038

276.7

24

3991

371.9

23

3988

280.2

25

Day 14

Mean

S.D.

N

4032 D

251.4

24

4050

240.5

24

4015

342.2

23

3971

266.6

25

Day 16

Mean

S.D.

N

4050 D

270.7

24

4090

286.9

24

4049

331.3

23

4009

291.3

25

Day 19

Mean

S.D.

N

4079 D

313.9

24

4106

323.2

24

4091

368.7

22

4036

308.3

25

Day 21

Mean

S.D.

N

4070 D

316.6

24

4102

325.8

24

4092

371.6

22

4030

324.9

25

Day 23

Mean

S.D.

N

4063 D

313.5

24

4095

279.6

24

4089

370.8

22

4022

333.6

25

Day 25

Mean

S.D.

N

4059 D

278.8

24

4078

270.3

24

4080

338.1

22

4004

334.0

25

Day 28

Mean

S.D.

N

4056 D

253.8

24

4094

275.6

24

4096

323.6

22

4048

300.1

24

Day 29

Mean

S.D.

N

4075 D

261.5

24

4105

275.2

24

4116

335.8

22

4060

294.9

24

 

D=Dunnett-test (two-sided)

 

Table 14: Mean maternal body weight change during gestation – grams

 

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Days 0 to 2

Mean

S.D.

N

83.2 D

46.80

24

76.3

42.51

24

75.0

41.52

23

64.5

45.09

25

Day 2 to 4

Mean

S.D.

N

58.6 D

34.98

24

59.5

26.74

24

54.3

23.97

23

46.3

52.87

25

Day 4 to 6

Mean

S.D.

N

30.6 D

37.49

24

41.0

71.01

24

46.0

32.71

23

38.1

76.64

25

Day 6 to 9

Mean

S.D.

N

82.4 D

52.25

24

76.7

63.04

24

67.5

78.12

23

56.8

58.10

25

Day 9 to 11

Mean

S.D.

N

44.6 D

34.67

24

45.5

44.56

24

47.5

40.96

23

45.7

54.88

25

Day 11 to 14

Mean

S.D.

N

22.9 D

89.41

24

12.1

95.44

24

24.1

104.33

23

-16.6

130.69

25

Day 14 to 16

Mean

S.D.

N

17.8 D

102.44

24

40.2

88.11

24

34.0

107.80

23

38.1

77.44

25

Day 16 to 19

Mean

S.D.

N

29.1 D

95.36

24

15.3

106.80

24

31.3

111.41

22

26.5

150.35

25

Day 19 to 21

Mean

S.D.

N

-9.0 D

56.80

24

-3.4

40.07

24

0.7

58.95

22

-5.3

50.45

25

Day 21 to 23

Mean

S.D.

N

-6.5 D

59.44

24

-6.8

61.58

24

-2.5

44.88

22

-8.7

41.68

25

Day 23 to 25

Mean

S.D.

N

-4.1 D

75.53

24

-17.0

42.96

24

-9.0

63.80

22

-17.7

60.66

25

Day 25 to 28

Mean

S.D.

N

-3.1 D

76.19

24

16.0

67.83

24

16.0

73.00

22

19.0

94.14

24

Day 28 to 29

Mean

S.D.

N

18.5 D

43.84

24

10.9

34.81

24

19.6

35.10

22

12.8

39.47

24

D=Dunnett-test (two-sided)

 

Table 15: Summary of mean maternal body weight change during gestation – grams

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Days 0 to 6

Mean

S.D.

N

172.4 D

80.18

24

176.7

85.03

24

175.3

69.82

23

149.0

79.67

25

Day 6 to 28

Mean

S.D.

N

174.1 D

146.69

24

178.5

182.42

24

210.9

180.61

22

162.5

212.08

24

Day 0 to 29

Mean

S.D.

N

365.0 D

164.25

24

366.1

232.16

24

410.0

207.24

22

321.5

205.70

24

D=Dunnett-test (two-sided)

 

Table 16: Mean gravid uterine weights and net maternal body weight change – grams

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Gravid uterus

Mean

S.D.

N

464.4 D

123.44

24

492.2

88.75

24

488.3

95.12

22

420.5

153.43

24

Carcass

Mean

S.D.

N

3610.2 D

282.67

24

3613.0

252.95

24

3627.3

288.58

22

3639.9

334.38

24

Net weight change from day 6

Mean

S.D.

N

-271.8 D

172.77

24

-302.8

167.46

24

-257.8

182.94

22

-245.3

236.39

24

D=Dunnett-test (two-sided)

Carcass weight = terminal body weight minus uterine weight

Net weight change from day 6 = carcass weight minus day 6 body weight

 

Table 17: Summary Reproductive data

 

 

Test group 0

Test group 1

Test group 2

Test group 3

 

 

0 mg/kg bw/d

50 mg/kg bw/d

150 mg/kg bw/d

500 mg/kg bw/d

Females Mated [N]

 

25

25

25

25

 

Pregnant [N]

24

24

23

25

 

Conception rate [%]

96

96

92

100

 

Aborted [N]

0

0

0

1

 

Premature Births [N]

0

0

0

0

 

Dams with viable fetuses [N]

24

24

22

24

 

Dams with all resorptions [N]

0

0

0

0

Female mortality [N (%)]

 

0 Fi (0)

0 (0)

1 (4.0)

1 (4.0)

Pregnant at terminal sacrifice [N (%)]

 

24 Fi (96)

24 (96)

22 (88)

24 (96)

Corpora Lutea [mean ± S.D. (total)]

 

10.9 D ± 2.41 (262)

11.3 ± 1.87 (270)

10.8 ± 2.17 (238)

10.0 ± 3.13 (240)

Implantation Sites [mean ± S.D. (total)]

 

10.0 D ± 3.21 (239)

10.5 ± 2.15 (251)

10.3 ± 2.06 (227)

9.3 ± 3.91 (222)

Preimplantation loss [mean ± S.D.]

 

10.4 D ± 20.98

7.3 ± 10.13

4.4 ± 2.83

12.3 ± 24.21

Postimplantation loss [mean ± S.D.]

 

4.5 D ± 8.21

7.2 ± 8.02

6.8 ± 8.37

6.9 ± 11.49

Resportions

 

 

 

 

 

 

Total [mean ± S.D. (total)]

0.5 D ± 0.93 (12)

0.8 ± 0.83 (19)

0.7 ± 0.78 (15)

0.9 ± 1.68 (21)

 

Total [mean % ± S.D.]

4.5 D ± 8.21

7.2 ± 8.02

6.8 ± 8.37

6.9 ± 11.49

 

Early [mean ± S.D. (total)]

0.4 D ± 0.58(9)

0.6 ± 0.77(15)

0.4 ± 0.73(8)

0.6 ± 0.97(15)

 

Early [mean % ± S.D.]

3.4 D ± 5.22

5.8 ± 7.73

3.6 ± 7.7

5.3 ± 7.92

 

Late [mean ± S.D. (total)]

0.1 D ± 0.45(3)

0.2 ± 0.38(4)

0.3 ± 0.57(7)

0.3 ± 0.9(6)

 

Late [mean % ± S.D.]

1.1 D ± 3.89

1.4 ± 3.19

3.1 ± 5.88

1.6 ± 5.55

Dead fetuses [N]

 

0

0

0

0

Live fetuses [mean ± S.D. (total)]

 

9.5 D ± 3.11 (227)

9.7 ± 1.97 (232)

9.6 ± 2.13(212)

8.4 ± 3.24(201)

Live fetuses [mean % ± S.D.]

 

95.5 D ± 8.21

92.8 ± 8.02

93.2 ± 8.37

93.1 ± 11.49

 

Females [mean ± S.D. (total)]

4.8 D ± 2.21(115)

4.9 ± 2.33(117)

4.9 ± 2.04(108)

4.3 ± 2.18(103)

 

Females [mean % ± S.D.]

45.1 D ± 19.09

45.2 ± 19.38

47.4 ± 15.48

46.3 ± 23.78

 

Males [mean ± S.D. (total)]

4.7 D ± 1.93(112)

4.8 ± 1.89(115)

4.7 ± 1.67(104)

4.1 ± 2.26(98)

 

Males [mean % ± S.D.]

50.4 D ± 19.94

47.6 ± 20.17

45.8 ± 12.06

46.7 ± 25.63

% live females

 

50.7

50.4

50.9

51.2

% live males

 

49.3

49.6

49.1

48.8

D=Dunnett-test (two-sided)

Fi=Fisher’s exact test (one-sided)

 

Table 18: Fetal weights

 

 

Test group 0

Test group 1

Test group 2

Test group 3

 

 

0 mg/kg bw/d

50 mg/kg bw/d

150 mg/kg bw/d

500 mg/kg bw/d

Fetal weights [unit: g]

 

 

 

 

 

 

All viable fetuses [mean ± S.D. (N)]

36 D ± 7.33(24)

35.6 ± 5.67(24)

35.6 ± 4.74(22)

36.6 ± 7.71

 

Females [mean ± S.D. (N)]

36.3 D ± 7.33(24)

35.3 ± 5.97(24)

36.2 ± 5.27(22)

35.6 ± 7.11(23)

 

Males [mean ± S.D. (N)]

34.1 D ± 5.59(22)

35.1 ± 5.06(23)

35.3 ± 4.83(22)

35.2 ± 6.32(22)

D=Dunnett-test (two-sided)

DISCUSSION

In a prenatal developmental toxicity study, cyclohexyl methacrylate was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28).

Analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution and the stability of the test substance in the vehicle.

Clinical observations revealed no toxicologically relevant difference between the animals receiving 50, 150 or 500 mg/kg bw/d cyclohexyl methacrylate and the controls.

The mean food consumption of the high-dose dams (500 mg/kg bw/d) was significantly below the concurrent control at the beginning of the treatment and recovered afterwards, however, overall these animals consumed about 6 % less food than the control group. There was no treatment related effect on body weights/body weight gain, carcass weights or corrected (net) body weight gain at all dose levels. The minor effect on food consumption with no consequence for the well-being of the animals is indicative of a slight local effect in the gastrointestinal tract of the animals rather than a sign of systemic toxicity.

No differences of toxicological relevance between the control and the treated groups (50, 150 or 500 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose.

Fetal examinations revealed no toxicologically relevant adverse effect of the compound on embryofetal development.

 

CONCLUSION

Under the conditions of this prenatal developmental toxicity study, the oral administration of cyclohexyl methacrylate to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused no evidence of systemic maternal toxicity up to the high- dose level of 500 mg/kg bw/d. The minor effect on food consumption with no consequence for the well-being of the animals at this dose is indicative of a slight local effect in the gastrointestinal tract of the animals rather than a sign of systemic toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 500 mg/kg bw/d.

Since there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is the highest dose of 500 mg/kg bw/d.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The study was conducted according to guideline and GLP.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
610 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The weight of evidence is considered appropriate and the quality of the data is sufficient for assessment.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

One oral prenatal developmental toxicity study in rabbits using the test substance cyclohexyl methacrylate is available. Also in the the combined OECD 408/422 test in rats no indications of a developmental toxicity were observed up to the highest dose of 1000 mg/kg bw. Furthermore a study with the lower methacrylate ester methyl methacrylate is used to support the assessment of potential developmental toxicity of cyclohexyl methacrylate.

 

For the inhalation route no studies on developmental toxicity of cyclohexyl methacrylate are available, therefore studies of the hydrolysis products cyclohexanol, methacrylic acid and the lower methacrylate ester methyl methacrylate were used to assess the potential of cyclohexyl methacrylate for developmental toxicity via inhalation and support the observations from the oral route.

 

Key study

Oral exposure

Cyclohexyl methacrylate

Cyclohexyl methacrylate was tested for its prenatal developmental toxicity in New Zealand White rabbits. The test substance was administered as an aqueous suspension to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 50, 150 and 500 mg/kg body weight/day on gestation days (GD) 6 through 28. The vehicle control group, consisting of 25 females, was dosed with the vehicle (drinking water with 10 mg/100 mL Cremophor EL) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 22-24 females per group had implantation sites.

Clinical observations revealed no toxicologically relevant difference between the animals receiving 50, 150 or 500 mg/kg bw/d Cyclohexyl methacrylate and the controls.

The mean food consumption of the high-dose dams (500 mg/kg bw/d) was significantly below the concurrent control at the beginning of the treatment and recovered afterwards, however, overall these animals consumed about 6 % less food than the control group. There was no treatment related effect on body weights/body weight gain, carcass weights or corrected (net) body weight gain at all dose levels. The minor effect on food consumption with no consequence for the well-being of the animals is indicative of a slight local effect in the gastrointestinal tract of the animals rather than a sign of systemic toxicity.

No differences of toxicological relevance between the control and the treated groups (50, 150 or 500 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. Fetal examinations revealed no toxicologically relevant adverse effect of the compound on embryofetal development.

During the prenatal developmental toxicity study in rabbits no evidence of systemic maternal toxicity up to the high- dose level of 500 mg/kg bw/d was observed. The minor effect on food consumption was indicative of a slight local effect in the gastrointestinal tract of the animals rather than a sign of systemic toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 500 mg/kg bw/d. No evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose was observed. Therefore the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is the highest dose of 500 mg/kg bw/d.

 

 

Supporting studies

Oral exposure

Cyclohexyl methacrylate, Modified Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (OECD 408/422), rat: NOAEL general, systemic toxicity:300 mg/kg bw/d based on functional liver impairment; NOAEL fertility and reproductive performance: 1000 mg/kg bw/d; NOAEL developmental toxicity: 1000 mg/kg bw/d (BASF SE 2018)

Methyl methacrylate

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

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

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

 

Inhalation exposure

Methyl methacrylate

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

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

 

Additionally, Rohm & Haas (Tansy et al., 1976) reported no signs of developmental toxicity in a study with mice, which were exposed to ca. 0.48 or 1.64 mg/L methyl methacrylate (corresponding to 116 and 400 ppm) for 6 hrs/day on GD 4-13.

 

Methacrylic acid

Furthermore a study equivalent to OECD guideline 414 was performed with the hydrolysis product methacrylic acid (Saillenfait et al., 1999). Groups of 19 to 25 pregnant female rats were whole body exposed for 6 hr/d, during gestation days 6 to 20 with concentrations of 0, 50, 100, 200, and 300 ppm (0, 179, 358, 716 and 1076 mg/m³). Overt maternal toxicity (decreased body weight and food consumption) was observed at 300 ppm. The treatment did not lead to embryo- or foetal lethality or foetal malformations at any concentration. Thus the NOAEL for developmental toxicity was set to the highest tested concentration, i.e. 300 ppm (1076 mg/m³).

 

Cyclohexanol

In the reproductive component of the modified inhalative OECD 422 guideline study described above for the hydrolysis product cyclohexanol, no test article-related effect was evident during the premating, gestation, or lactation periods on body weight, reproductive and fertility parameters, and litter size data (US EPA, 2010). Pup sex ratios and pup survival to lactation day 4 were also unaffected by treatment. Two of the 11 pregnancies (18.2 %) in the high dose group resulted in no viable pups at parturition.

Further, lower mean pup body weights were seen at birth for the high dose group. Female adult rats showed no significant adverse clinical signs and no teratological effects were observed at any exposure level. At the highest exposure level, treatment-related effects were a slight increased incidence of pregnancies with no viable fetuses and lower pup weights. The NOAEC for developmental toxicity was set to 0.61 (150 ppm) mg/L.

 

Conclusions

Based on the prenatal developmental toxicity study in rabbits with Cyclohexyl methacrylate the no observed adverse effect level (NOAEL) for maternal toxicity was determined to be 500 mg/kg bw/d. As no toxicologically relevant adverse effects of the test substance on fetal morphology were detected, the NOAEL for prenatal developmental toxicity was established to be 500 mg/kg bw/d.

 

The result of the oral study conducted with the test substance is also supported by the available oral study conducted with Methyl methacrylate. This study underlines the absence of developmental effects, as pups of rabbits treated orally in the 2-generation study, did not show any adverse effects on F1 and F2 generation up to the highest tested concentration of 400 mg/kg bw/d.

Furthermore inhalation studies on rats and mice support the results, as no developmental effects were observed up to 610 mg/L with the hydrolysis products cyclohexanol and methacrylic acid as well as the read across substance methyl methacrylate. Even at overt maternal toxicity, no teratogenic effects were observed in rat pups.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. In several reliable studies of the analogous substance methyl methacrylate and the hydrolysis products cyclohexanol and methacrylic acid in rats, rabbits and mice, no effects on fertility or developmental toxicity were observed, even in maternal toxic doses. Therefore, cyclohexyl methacrylate does not need to be classified as reproductive or developmental toxicant according to Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.