Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
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

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

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.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

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.

Applicant's summary and conclusion