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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
06 Aug 2012 - 27 Feb 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
27 July 1995
Deviations:
yes
Remarks:
20 animals per sex were used.
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, July 2000
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: orange solid
- Analytical purity: > 99%
- Expiration date of the lot/batch: unlimited
- 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: (P) 1 - 11 wks
- Housing: individually in Makrolon type M III cages, except during mating and postnatal days 0-4
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum

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
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, deionized 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.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: single-housing
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 as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany, under the responsibility of a 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 deionized water for a period of 5 days at room temperature was proven before the start of the study.

Homogeneity analyses of the test substance preparations were performed in samples of the highest and lowest concentrations at the start of the administration period. These samples also served for concentration control.
Duration of treatment / exposure:
40 to 55 days
males: 2 weeks premating and during mating
females: 2 weeks premating, during mating, gestation and 4 days lactation
Frequency of treatment:
daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: limit test with 1000 mg/kg bw was done based on unexpected total implantation loss at that dose in another OECD 421 study (80R0323/08X008)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day during work days and once on weekends

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule: twice a day during work days and once on weekends

BODY WEIGHT: Yes
- Time schedule for examinations: males: on the first day of dosing and weekly thereafter; females: on the first day of dosing and once weekly during the premating period, on days 0, 7, 14 and 20 of gestation and during lactation on the same day as litters (days 0 and 4 postnatal)

FOOD CONSUMPTION:
- Time schedule: weekly during premating, during gestation and lactation periods on days 0, 7, 14 and 20 (gestation) and 0 and 4 (lactation), no food consumption was determined for males during mating and postmating periods and for females without positive evidence of sperm and without litter
Litter observations:
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
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: after mating
- Maternal animals: All surviving animals on day 4 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were weighed: testes and epididymides. Furthermore, the following tissues were prepared for microscopic examination: all gross lesions, testes, epididymides, ovaries, uterus and oviducts, prostate, seminal vesicles and coagulating gland. Full histopathology of ovaries, testes and epididymides was performed.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic and abdominal cavities.
Statistics:
For the hypothesis of equal means student's t-test (two-sided) was performed. For the hypothesis of equal proportions Fisher's exact test (one-sided) was applied. For the hypothesis of equal medians one-sided Wilcoxon test was performed. Results were found to be significant at p<0.05.
Reproductive indices:
Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100
(*defined by a female with vaginal sperm or with implants in utero)
Male fertility index (%) = (number of males proving their fertility*/number of males placed with females) x 100
(*defined by a female with implants in utero)
Female mating index (%) = (number of females mated*/number of females placed with males) x 100
(*defined as the number of females with vaginal sperm or with implants in utero)
Female fertility index (%) = (number of females pregnant*/number of females mated**) x 100
(*defined as the number of females with implants in utero; **defined as the number of females with vaginal sperm or with implants in utero)
Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
(*defined as the number of females with implants in utero)
Offspring viability indices:
Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100
Post implanatation loss (%) = (number of implantations - number of pups delivered/number of implantations) x 100
Viability index (%) = (number of live pups on day 4 after birth/number of live pups on the day of birth) x 100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no mortalities observed during the study period. All male and female animals of test group 1 (1000 mg/kg bw/d) showed orange discolored feces from study day 2 until the end of the study. Two sperm positive females of test group 1 (Nos. 128 and 133 – 1000 mg/kg bw/d) did not deliver F1 pups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption of the male and female F0 generation parental animals in test group 1 (1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation. Mean body weights and mean body weight gain of the F0 males and females in test group 1 (1000 mg/kg bw/d) were comparable to the concurrent control group throughout the entire study period.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The male mating index was 85% in control group and 100% in test group 1 (1000 mg/kg bw/d). This finding 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.
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter.
Three males of control group and two males of test group 1 did not generate F1 pups with paired females.
Thus, the male fertility index was 85% in the control group and 90% in test group 1. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
The female mating index calculated after the mating period for F1 litter was 85% in the control group and 100% in test group 1.
The mean duration until sperm was detected (GD 0) varied between 3.4 in test group 0 and 3.7 days (in test group 1).
All sperm positive rats delivered pups or had implants in utero with the following exception:
• Control group female Nos. 101, 116 and 117 (mated with male No. 1, 16 and 17) did not become pregnant.
• high-dose females Nos. 128 and 133 (mated with males Nos. 28 and 33) did not become pregnant.
The female fertility index was 100% in the control group and 90% in test group 1. 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 (22.0 days in test group 1 and 22.3 days in the control group).
The gestation index was 100% in all test groups.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (11.2 and 13.0 implants/dam in all test groups (0, and 1000 mg/kg body weight/day)). There were no statistically significant differences in post-implantation loss between the groups (6.5% / 5.1%), and the mean number of F1 pups delivered per dam remained unaffected (10.4 and 12.3 pups/dam at 0 and 1000 mg/kg bw/d).

ORGAN WEIGHTS (PARENTAL ANIMALS)
All mean absolute or relative weight parameters did not show significant differences when compared to the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
A treatment-related yellow discoloration of contents was observed in the glandular stomach, jejunum, cecum, and the colon.

HISTOPATHOLOGY (PARENTAL ANIMALS)
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.
One male animal (No. 1, control group) showed a severe (grade 4) multifocal degeneration of the testes resulting in an aspermia of the epididymides, correlating with the gross finding size reduction. These findings explain why the female animal (No. 101) was not pregnant. All other females that were not pregnant as well as their male mating partners did not show relevant findings in the investigated organs. The non-pregnant female rats of the control group (Nos. 108 and 115) exhibited a cyst in the right oviduct as well as a dilated uterus and vagina. High-dose female No. 132 (1000 mg/kg bw/d) had a dilated uterus at gross necropsy. None of the other non-pregnant females (Nos. 115, 120, 125 and 134) had any relevant gross lesions.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
systemic/reproductive performance/fertility
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 effects observed at the limit dose

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The viability index indicating pup mortality during lactation (PND 0 - 4) was 96.5% in the control group and 99.6% in test group 1. Due to the lack of dose response relationship this was assessed as being incidental. 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.

CLINICAL SIGNS (OFFSPRING)
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

BODY WEIGHT (OFFSPRING)
Body weight of female pups in test group 1 was slightly decreased on day 4, but the overall mean pup weight of both sexes was comparable to the control group. Thus, this finding was assessed as being incidental. One male runt was seen in test group 0. One male and five female runts were seen in test group 1 (1000 mg/kg bw/d). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. 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.

GROSS PATHOLOGY (OFFSPRING)
Respectively one male F1 pup of control group showed an empty stomach. One female F1 pup of test group 1 (1000 mg/kg bw/d) showed post mortem autolysis. One female pup of test group 1 (1000 mg/kg bw/d) was cannibalized. This finding was assessed as being spontaneous in nature and without biological relevance.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
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 effects observed (as far as F1 can be assessed in a screening study)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion