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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: 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
Limit test:
no

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
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:
Perylen Schwarz I 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.
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 drinking water for a period of 7 days at room temperature was proven before the start of the study (Study No. 08L00232).
Homogeneity analyses of the test substance preparations were performed in samples of the highest and lowest concentrations at the start of the administration period. In samples of the middle concentration only concentration control analyses were performed.
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".
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

Maternal 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 AND COMPOUND INTAKE (if feeding study):
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).
Fetal examinations:
- External examinations: Yes
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
Indices:
mating index, fertility index, gestation index, postimplantation loss, live birth index, pup number and status at delivery, viability index

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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
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
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

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The mean duration of gestation was similar in all test groups (i.e. between 21.7 and 22.2 days).
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Implantation and delivery were not affected by treatment, since neither the mean number of implantation sites nor the average litter size showed any statistically significant differences between the groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): 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. These findings were incidental and regarded to be spontaneous in nature.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Changes in sex ratio:
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.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean number of delivered F1 pups per dam was evenly distributed about the groups.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
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.
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
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.
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.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no teratogenic effects, no developmental toxicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental 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 developmental toxicity was concluded to be 1000 mg/kg bw/day for maternal animals and offspring. The test substance did not show abnormalities in offspring and thus, was not considered to be teratogenic.
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 CO2and 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 NOAEL for developmental toxicity in the F1 progeny was found to be 1000 mg/kg bw/d. The NOAEL for general, systemic toxicity was 1000 mg/kg bw/d.