Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
During pup necropsy observation, two pups of female No.128 (PND 4; test group 2) and each 8 pups of females Nos. 103, 110, 113, 115 (PND 13; control and test group 1) were inadvertently not documented. This does not harm the validity of the study.
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (Jul 2000)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperatures

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany GmbH
- Age at study initiation: About 11 - 12 weeks (male animals) and about 10 weeks (female animals)
- Weight at study initiation: 160-250g
- Housing: During the study period, the rats were housed individually in polycarbonate cages type III (floor area of about 800 cm²), with the following exceptions:
• During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm² (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany
• During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III
- Diet: ad libitum, ground Kliba maintenance diet mouse-rat “GLP"
- Water: ad libitum
- Acclimation period: 20d
- Fasting: 16h before administration

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil (which was heated up to 70°C) was filled up to the desired volume and subsequently released with a magnetic stirrer. Onwards, the suspension was heated up to 80°C. The test substance preparations were produced weekly, at least.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 2 weeks
- Proof of pregnancy: vaginal smear was sperm positive, or a copulation plug was observed referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): pregnant animals and litter together
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, one sample from the mid concentration was additionally taken for concentration control analysis.
The stability of of the test itme in corn oil was demonstrated over a period of 7 days at room temperature. As the test substance preparations were not stored longer than this time period, the stability was guaranteed. Considering the results in the homogeneity analysis, it can be concluded that the test item was distributed homogeneously in corn oil. Regarding the mean values, the concentrations of the test item in corn oil were found to be in the range of 90-110% of the nominal concentration. These results demonstrated the correctness of the concentrations of the test substance in corn oil.
Duration of treatment / exposure:
The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 28d study

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as
well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal. The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
• During the mating period, the 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 of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume)
• Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of the administration volume)

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd 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 for GD 7, 14 and 20.
• Food consumption of the females which gave birth to a litter was determined for PND 4, 7, 10 and 13.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

CLINICAL PATHOLOGY
Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4 and TSH).
Oestrous cyclicity (parental animals):
Estrous cycle determination prior to treatment was performed in a pool of up to 50 non-randomized female animals. Only animals with regular estrous cycle were selected for randomization before the start of the treatment period.
In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
Litter observations:
STANDARDISATION OF LITTERS
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups per litter were sacrificed. 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. The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements was done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth.

All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.


BODY WEIGHT
The pups were weighed on the day after birth (PND 1) as well as on PNDs 4, 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). In the summary tables pup body weights and pup body weight change are listed for males, females and males + females. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh
less than 75% of the mean weight of the respective control pups.

GROSS EXAMINATION OF DEAD PUPS:
All stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
Postmortem examinations (parental animals):
SACRIFICE & GROSS NECROPSY
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Ovaries
4. Prostate
5. Seminal vesicles with coagulating glands
6. Testes
7. Thyroid glands (fixed)
8. Uterus (with cervix)

ORGAN FIXATION
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Cervix
3. Coagulating glands
4. Epididymides (modified Davidson’s solution)
5. Ovaries (modified Davidson’s solution)
6. Oviducts
7. Prostate gland
8. Seminal vesicles
9. Testes (modified Davidson’s solution)
10.Thyroid glands
11.Vagina
12.Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E (1964))

HISTOPATHOLOGY
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:

Test group 0 1 2 3
Testes A1 A3 A3 A1
Epididymides A1 A3 A3 A1
Ovaries A1 A1 A1 A1
Uterus Uterus with cervix and vagina A1 A1 A1 A1

A = Hematoxylin and Eosin (H&E) stain
1 = All animals/test group
3 = Mating pairs suspected of reduced fertility
Postmortem examinations (offspring):
SACRIFICE & GROSS NECROPSY
On post-natal day (PND) 4, as a result of standardization, surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, these pups were examined externally, eviscerated and their 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 (T4 and TSH). Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Laboratory Pathology for possible further processing. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. All culled pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and visceral) examination. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a case by case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.
Statistics:
- DUNNETT-test (two-sided) for the hypothesis of equal means: Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index
- FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: Male and female mating indices, male and female fertility indices, females
mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups
- WILCOXON test (onesided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
- WILCOXON test (onesided-) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians: Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival Index
- WILCOXON test (two-sided) for the hypothesis of equal medians: % live male day x, % live female day x
- KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the equal medians: Number of cycles and Cycle Length, Blood parameters, Weight parameters
Reproductive indices:
Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index, Live birth index, Post implantation loss
Offspring viability indices:
Viability Index, Survival Index, Sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Reddish smeared fur in mouth region within 2 hours after the administration was observed in 7 of 10 males of test group 3 (1000 mg/kg bw/d) and in 1 of 10 males of test group 2 (300 mg/kg bw/d) during pre-mating and in 3 of 10 males of test group 3 (1000 mg/kg bw/d) during mating. Salivation within 2 hours after the administration was observed in all males and 8 of 10 females of test group 3 (1000 mg/kg bw/d) and in 4 of 10 males of test group 2 (300 mg/kg bw/d) during pre-mating as well as in all males and all females of test group 3 (1000 mg/kg bw/d) and in 2 of 10 males of test group 2 (300 mg/kg bw/d) during mating. These findings were considered to be related to treatment but not assessed as adverse and toxicologically relevant effects.
During gestation, reddish smeared fur in mouth region within 2 hours after the administration occurred in 1 of 10 females of test group 3 (1000 mg/kg bw/d) during gestation. Salivation within 2 hours after the administration was observed occurred in all females of test group 3 (1000 mg/kg bw/d). These findings were considered to be related to treatment but not assessed as adverse and toxicologically relevant effects.
During lactation, reddish smeared fur in mouth region within 2 hours after the administration occurred in 1 of 10 females of test group 3 (1000 mg/kg bw/d) during lactation. Salivation within 2 hours after the administration was observed in 8 of 10 females of test group 3 (1000 mg/kg bw/d). These findings were considered to be related to treatment but not assessed as an adverse and toxicologically relevant effect.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related changes in mean body weights and body weight change values were observed for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related changes in food consumption were observed.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related changes in water consumption were observed.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Description (incidence and severity):
No treatment-related changes of T4 and TSH hormone levels occurred in parental males of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d).
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were observed in the parental male and female animals. Also, no findings were observed to explain the increased uterus weights of females of test groups 1, 2 and 3.
The female animals (Nos. 114, 117, 118, 119, 120, 125, 133, 135) and the male mating partners (Nos. 14, 17, 18, 19, 20, 25, 33, 35) showed no histopathological findings that could explain the impaired fertility.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data revealed regular cycles in the F0 females of all test groups including the control. The mean estrous cycle duration in the different test groups was 3.9 days in test groups 0 to 3 (control, 100, 300 and 1000 mg/kg bw/d, respectively).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The stages of spermatogenesis in the testes of male animals in test group 3 were comparable to those of the controls.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
- Male mating index
The male mating index calculated after the mating period for producing F1 litter was 100% in test groups 0 to 3 (control, 100, 300 and 1000 mg/kg bw/d, respectively).

- Male fertility index
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Male animal Nos. 14, 17, 18, 19, 20 of test group 1 (100 mg/kg bw/d), which were mated with
female animal Nos. 114, 117, 118, 119, 120, male animal No. 25 of test group 2 (300 mg/kg bw/d), which was mated with female animal No. 125, and male animal Nos. 33, 35 of test group 3 (1000 mg/kg bw/d), which were mated with female animal Nos. 133, 135, did not generate F1 pups. Thus, the male fertility index was 100% in test group 0 (control, 0 mg/kg bw/d), 50% in test group 1 (100 mg/kg bw/d), 90% in test group 2 (300 mg/kg bw/d) and 80% in test group 3 (1000 mg/kg bw/d). The values for test groups 0, 2 and 3 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. Although the index for male animals of test group 1 was only 50%, a relation to treatment was not considered since no dose-response relationship occurred.

- Female mating index
The female mating index calculated after the mating period for producing F1 litter was 100% in test groups 0 to 3 (control, 100, 300 and 1000 mg/kg bw/d, respectively). The mean duration until sperm was detected (GD 0) was 2.6 days for test group 0, 1.7 days for test group 1, 3.6 days for test group 2 and 2.8 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

- Female fertility index
Most sperm positive rats delivered pups with the exception of female animal Nos. 114, 117, 118, 119, 120, which were mated with male animal Nos. 14, 17, 18, 19, 20 of test group 1 (100 mg/kg bw/d), female animal No. 125, which was mated with male animal No. 25 of test group 2 (300 mg/kg bw/d), and female animal Nos. 133, 135, which were mated with male animal Nos. 33, 35 of test group 3 (1000 mg/kg bw/d), had sperm in vaginal smear but delivered no pups and showed no implants. Thus, the female fertility index was 100% in test group 0 (control, 0 mg/kg bw/d), 50% in test group 1 (100 mg/kg bw/d) and 90% in test group 2 (300 mg/kg bw/d) and 80% in test group 3 (1000 mg/kg bw/d) The values for test groups 0, 2 and 3 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. Although the index for female animals of test group 1 was only 50%, a relation to treatment was not considered since no dose-response relationship occurred.
The mean duration of gestation was similar in all test groups, i.e. 22.4 days in test group 0, 22.6 days in test group 1, 22.0 days in test group 2, and 22.2 days in test group 3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

- Gestation index
The gestation index was 100% in all test groups.

- Live birth indices
The rate live birth indices were 100% in all test groups.

- Postimplantation loss
The postimplantation loss was 6.8% in test group 0 (control), 9.5% in test group 1 (100 mg/kg bw/d), 5.0% in test group 2 (300 mg/kg bw/d) and 4.3% in test group 3 (1000 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. A treatment-related increase in postimplantation loss was not observed in any test group.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse findings reported

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All F1 pups of any test group (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4 or PND 13. Discolored skin was observed in one male pup of test group 1 (100 mg/kg bw/d) on PND 3-5 and one male pup of test group 2 (300 mg/kg bw/d) on PND 0-1. The findings were regarded to be incidental.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- Pup number and status at delivery
The mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3. No significant deviations occurred.

- Pup viability index/mortality
The viability index indicating pup mortality during PND 0-4 was 100.0% in test group 0 (control), 98.5% in test group 1 (100 mg/kg bw/d), 99.1% in test group 2 (300 mg/kg bw/d) and 100% in test group 3 (1000 mg/kg bw/d).
The survival index indicating pup mortality during PND 4-13 was 100.0% in test groups 0, 1 and 3, and 96.7% in test group 2. As no dose-response relationship occurred a relation to treatment was excluded.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group. One male and 2 female runts were seen in test group 0 (control group; 0 mg/kg bw/d), one male runt was seen in test group 1 (100 mg/kg bw/d), 5 male and 3 female runts were seen in test group 2 (300 mg/kg bw/d) and 3 male and 5 female runts were seen in test group 3 (1000 mg/kg bw/d) on PND 1. All values were within the range of the biological variation inherent in the strain of rats used for this study. For F1 male pups in test group 2 (300 mg/kg bw/d), the mean body weights on PND 1 and PND 4 were significantly lower compared to the control. However, the weights did not differ on PND 7 and 13. Since no dose-response relationship occurred and the mean weights did not differ significantly from PND 7 onwards, a relation to treatment was excluded.
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
Description (incidence and severity):
No treatment-related changes of T4 and TSH hormone levels occurred in F1 female pups of test groups 11, 12 and 13 (100, 300 and 1000 mg/kg bw/d) on PND 13.
On PND 13, T4 values were significantly lower in F1 male pups of test groups 12 and 13 (300 and 1000 mg/kg bw/d) when compared to controls. However, T4 means were within the historical control range (males, PND13 43.48-75.77 nmol/L). TSH values were not changed. Therefore, the T4 decrease in these male pups were regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related findings were observed.
Histopathological findings:
no effects observed
Description (incidence and severity):
On PND 13, the thyroid glands of male animals in test groups 1, 2, and 3 were comparable to those of the control group.
Other effects:
no effects observed
Description (incidence and severity):
- 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 distance was significantly increased in F1 male animals of test groups 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d). Anogenital index was significantly increased in F1 male animals of test groups 1-3 (100, 300
and 1000 mg/kg bw/d) and in F1 female animals of test groups 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d). The deviations to the control values were not assessed as related to treatment since all values were well within the historical control data. These values reflected the normal range of biological variation inherent in the strain of rats used for this study.

- 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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse findings reported

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test article to Wistar rats revealed no signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.
Executive summary:

The test item was administered by gavage to groups of 10 male and 10 female Wistar rats (F0 generation) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Corn oil served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7,14 and 20 and lactation days 4, 7, 10 and 13. In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1 after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted. At necropsy on PNDs 4 and 13, all pups were sacrificed with CO2 under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus 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. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing. 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. In parental animals of all ose groups, no treatment-related, adverse effects were observed regarding Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology. No treatment-related, adverse effects were observed. in F1 pups of all dose groups. Under the conditions of this Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test article to Wistar rats revealed no signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.