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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:
30 Aug 2011 - Feb 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test (Jul 2000)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid, powder, black
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 weeks (males/females)
- Weight at study initiation: average weights: 322 g (males); 201.6 g (females)
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions: During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, from water bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 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:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week. The administration volume was 10 mL/kg body weight.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out at the Competence Center Analytics Department of BASF SE, Ludwigshafen, Germany under the responsibility of the Study Director of this test facility. The study was carried out in compliance with the Principles of Good Laboratory Practice.
The stability of the test substance in water was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test material was distributed homogeneously in drinking water. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 99-111% of the nominal concentrations. These results demonstrate the correctness of the
concentrations.
Duration of treatment / exposure:
The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation 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

Examinations

Parental animals: Observations and examinations:
MORTALITY
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.

CLINICAL OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of littering was considered 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 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 of parturition (PND 0) and on PND 4.
• Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume. Therefore, these values are only documented in the Individual Tables (PART II).

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 during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 4.
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).
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Epididymides weights, testes weights, staging of spermatogenesis
Litter observations:
Viability/mortality:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.

Sex ratio:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

Clinical observations:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.

Pup body weight data:
The pups were weighed on the day after birth (PND 1) and on PND 4. 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).
“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.
Postmortem examinations (parental animals):
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. Testes

Histopathology:
The following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution:
All gross lesions, Cervix, Coagulating gland, Epididymides (modified Davidson’s solution), Ovaries (modified Davidson’s solution), Oviducts, Prostate gland, Seminal vesicles, Testes (modified Davidson’s solution), Vagina, Uterus.
Fixation was followed by histotechnical processing and examination by light microscopy. The following tissues were examined: testes, epididymides, ovaries from all control and high dose animals.
Postmortem examinations (offspring):
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
All pups were discarded after their evaluation.
Statistics:
The following statistical methods were used:
- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions.
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Non-parametric one-way analysis using KRUSKAL-WALLIS 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 (two-sided) for the equal medians
Reproductive indices:
male and female mating index, male and female fertility index, gestation index, postimplantation loss
Offspring viability indices:
Live birth index, pup number and status at delivery, viability index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All male animals of test group 2 (300 mg/kg bw/d) from study week 2 onwards and all male and female animals of test group 3 (1000 mg/kg bw/d) from study week 0 onwards showed black discolored feces. During gestation all animals of test groups 2 and 3 (300 and 1000 mg/kg bw/d) showed black discolored feces. All animals test groups 2 and 3 (300 and 1000 mg/kg bw/d) showed black discolored feces during lactation period.
Mortality:
no mortality observed
Description (incidence):
Female animal No. 105 of test group 0 (0 mg/kg bw/d) was found dead on study day 24 (GD 10).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No changes of toxicological concern with regard to body weight parameters of male animals were observed during the entire study period. During gestation body weight of test group 2 (300 mg/kg bw/d) was significantly increased on GD 20. Body weight change values of female animals of test groups 1 and 2 (100 and 300 mg/kg bw/d) were significantly increased from gestation day 14 to 20. Due to the lack of a dose response relationship this was assessed as being incidental.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No changes of toxicological concern with regard to food consumption of female animals were observed during 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):
None of the mean absolute or relative weight parameters showed significant differences when compared to the control group 0.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Black discoloration of the intestinal tract was noted in many animals of test group 2 (300 mg/kg bw/day) and 3 (1000 mg/kg bw/day). 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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All 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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The mean duration until sperm was detected (GD 0) was 2.6, 2.5, 2.7 and 2.1 days in test groups 0-3. All sperm positive rats delivered pup.
Reproductive performance:
no effects observed
Description (incidence and severity):
For F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index was 100% in all test groups. Fertility was proven for all of the F0 parental males within the scheduled mating interval to produce F1 litter. The male fertility index was 100%.
The female mating index calculated after the mating period for F1 litter was 100% for all test groups. The female fertility index was 100%.
The gestation index was 100% in test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) and 90% in test group 0 (0 mg/kg bw/d), due to an interim death of one animal during gestation without any clinical signs. This finding was incidental and regarded to be spontaneous in nature. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
Implantation, postimplantation loss and delivery were not affected by the treatment, since neither the mean number of implantation sites nor the average litter size showed any statistically significant differences between the groups.

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 test substance-related, adverse findings were noted.

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
Mortality / viability:
no mortality observed
Description (incidence and severity):
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% (test groups 0 and 1), 99% (test group 2) and 96% (test group 3). Two stillborn pups were seen in test group 2 and five stillborn pups in test group 3. The live birth index was in the normal range of the rat strain used and was assessed as being incidental.
The mean number of delivered F1 pups per dam was evenly distributed about the groups.
The two stillborn pups in test group 2 (300 mg/kg bw/d) and five stillborn pups in test group 3 (1000 mg/kg bw/d) were incidental and in the normal range of biological variation inherent in the strain of rats used for this study.
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 97% (test group 1), 98% (test group 2 and 3) and 100% (test group 0) and was in the normal range of biological variation inherent in the strain of rats used for this study.
Body weight and weight changes:
no effects observed
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.
In test group 0 and 2 (0 and 300 mg/kg bw/d) one male runt was seen, in test group 1 (100 mg/kg bw/d) one male and one female runt were seen and in test group 3 (1000 mg/kg bw/d) two male and two female runts were seen. This was within the normal range of the biological variation inherent in the strain of rats used for this study.
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:
effects observed, non-treatment-related
Description (incidence and severity):
One dead pup of test group 1 (100 mg/kg bw/d) and two stillborn pups of test group 3 (1000 mg/kg bw/d) showed post mortem autolysis.
This finding was assessed as being spontaneous in nature and without biological relevance.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups. Slight differences were regarded to be spontaneous in nature.

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 test substance-related, adverse findings were noted.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats.
Executive summary:

The test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 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). Drinking water served as vehicle. The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females. After 2 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 was detected in the vaginal smear. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings. 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. No treatment related parental toxicity was observed at any dose level. No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, macroscopic examination, organ weights, and microscopic examination). No treatment related reproduction toxicity was observed at any dose level. No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices). In conclusion, treatment with the test substance by oral gavage in male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg bw/day revealed no parental and reproduction toxicity up to 300 mg/kg bw/day. Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 1000mg/kg bw/d for the F0 parental rats. The NOAEL for general, systemic toxicity was 1000 mg/kg bw/d.