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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:
experimental study
Adequacy of study:
key study
Study period:
2011 - 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant study with well characterized material.
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid/violet to dark blue
- Analytical purity: 96.84 %
- Expiration date of the lot/batch: 04. Feb. 2021
- Stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor. The stability of the test substance in drinking water at room temperature over a period of 7 days and the homogenous distribution of the test substance in drinking water was confirmed by analysis.
- 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 - 12 weeks (males/females)- Weight at study initiation: mean (males): 304 g; mean (females): 201 g- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany, 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) with nesting material, for motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany and small amounts of bedding material.- Diet: Ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum- Water: drinking water (from water bottles); ad libitum- Acclimation period: 6 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20 - 24°C - Humidity (%): 30 - 70% - Air changes (per hr): 15/hr- 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.VEHICLE- Concentration in vehicle: 1 (100 mg/kg bw), 3 (300 mg/kg bw), 10 g/100 mL (1000 mg/kg bw)
Details on mating procedure:
- M/F ratio per cage: 1/1- Length of cohabitation: 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 (Mating attempts were performed for a maximum of 2 weeks). - Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy- After successful mating each pregnant female was caged (how): Pregnant animals and their litters were housed together until PND 4 (end of lactation). Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses confirmed the stability of the test substance in drinking water at room temperature over a period of 7 days, the homogeneous distribution of the test substance in drinking water, and the correctness of the prepared concentrations of the test substance preparations in drinking water (the values were in the expected range of the target concentrations, i.e. were always in a range of about 102-109% of the nominal concentrations).
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice (males: 34 / 35 days; females: 49 days)
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12 - 14 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:100, 300 and 1000 mg/kg bwBasis:actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: 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. 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.DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: Before initial test substance administration and thereafter at weekly intervals.BODY WEIGHT: Yes - Time schedule for examinations: Body weights of F0 parents were determined on study day 0 (start of the administration period) and thereafter 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.FOOD CONSUMPTION AND COMPOUND INTAKE:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: NoFood 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).WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): NoOTHER: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.
Sperm parameters (parental animals):
Parameters examined in male parental generation:testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS- Performed on day 4 postpartum: noPARAMETERS EXAMINEDThe 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 abnormalitiesGROSS EXAMINATION OF DEAD PUPS:yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE- Male animals: all surviving animals - Maternal animals: all surviving animals GROSS NECROPSY- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.HISTOPATHOLOGY / ORGAN WEIGHTS:Weight assessment was carried out on all animals. The following weights were determined:Adrenal glands, Anesthetized animals, Brain, Epididymides, Heart, Kidneys, Liver, Testes, Spleen, ThymusThe following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:Adrenal glands, All gross lesions, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve (modified Davidson’s solution), Esophagus, Extraorbital lacrimal gland, Epididymides (modified Davidson’s solution), Female mammary gland, Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs, Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina;From the liver, each one slices of the lobus dexter medialis and the lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast.
Postmortem examinations (offspring):
SACRIFICE- 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.- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:GROSS NECROPSY- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means; Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions; Proportions of affected pups per litter with necropsy observations: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians;
Reproductive indices:
Male / female mating indices, male / female fertility indices, gestation index, live birth index, postimplantation loss
Offspring viability indices:
Viability index was calculated using the number of live pups/litter on the day after birth, and on lactation day 4.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
females of the high dose group during gestation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
females of the high dose group during gestation
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No animal died prematurely in the present study. No test substance-related, adverse findings were noted. All animals of test group 1 (100 mg/kg bw/d) from study week 1 onwards and all animals of test groups 2-3 (300 and 1000 mg/kg bw/d) from study week 0 onwards showed blue discolored feces. During gestation all animals of test group 1 and 3 (100 and 1000 mg/kg bw/d) and 9 animals of test group 2 (300 mg/kg bw/d) showed blue discolored feces during gestation. All animals showed blue discolored feces during the lactation period.BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): In males of test group 1 (100 mg/kg bw/d) food consumption was decreased from study week 1 to 2. Significantly decreased (up to 87%) food consumption during the entire gestation period in females of test group 3 (1000 mg/kg bw/d).REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): In a single female of test group 2 (No. 126; 300 mg/kg bw/d) no sperm in vaginal smear was detected during the entire mating period. Due to the lack of a dose response relationship this was assessed as being incidental. One sperm-positive F0 female of the test group 0 (No. 103), 1 sperm-positive F0 female of test group 1 (No. 120) and 3 sperm-positive F0 female of test group 3 (Nos. 132, 133, 138) did not deliver any F1 pups. Female animals Nos. 103, 120, 132 and 133, which delivered no pups, showed no implantation sites. ORGAN WEIGHTS (PARENTAL ANIMALS): There were no effects on organ weights.GROSS PATHOLOGY (PARENTAL ANIMALS): No test substance-related, adverse findings were noted. The only findings were: Discoloration of contents of glandular stomach, jejunum, colon and discoloration of the tongue. These findings are regarded to be treatment related but not adverse. The macroscopically observed dilation of the uterus in three animals of test group 3 (1000 mg/kg bw) are regarded to be related to the cycle and therefore no pathologic finding.Fertility: Examination of males with no offsprings and of not pregnant females: Animal No 3 of test group 0 (0 mg/kg bw) showed an enlarged testis with glassy-transparent appearance. All other females that were not pregnant and all other male mating partners did not show gross lesions in reproduction relevant organs.HISTOPATHOLOGY (PARENTAL ANIMALS): No signs of toxicity in the respective tissues were noted. The only findings were: Discoloration of contents of glandular stomach and jejunum, blue particles on surface of forestomach, cecum, rectum and discoloration of the tongue. These findings are regarded to be treatment related but not adverse. In the forestomach of all investigated animals bluish particles were located on top of the mucosal surface. The mucosa did not show any additional findings.Fertility: One male animal of the control group (animal No 3) of which the female mating partner was not pregnant revealed in both testes diffuse tubular degeneration. In the epididymides of this animal aspermia or oligospermia were present. These findings were regarded to be responsible for the infertility observed. All other organs of the reproduction tract did not show any relevant finding. All investigated not pregnant females as well as the remaining male mating partners did not show relevant histopathological findings that could explain infertility.OTHER FINDINGS (PARENTAL ANIMALS):Male mating index: 100% in test groups 0, 1 and 3 and 90% in test group 2;Male fertility Index: 90% in test groups 0, 1 and 2 and 80% in test group 3;Female mating index: 100% in test groups 0, 1 and 3 and 90% in test group 2; Female fertility Index: 90% in test groups 0 and 1, 100% in test group 2 and 80% in test group 3;Gestation index: 100% in test groups 0, 1 and 2 and 88% in test group 3;

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive performance and fertility
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At highest dose tested no adverse effects observed.
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on reduced food consumption and decreased body weight gain during gestation at 1000 mg/kg bw.
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: At highest dose tested no adverse effects observed.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING): The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 99% (test group 0 and 1) and 100% (test group 2 and 3).CLINICAL SIGNS (OFFSPRING): No test substance-related, adverse findings were noted.BODY WEIGHT (OFFSPRING): Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group. GROSS PATHOLOGY (OFFSPRING): No test substance-related, adverse findings were noted.OTHER FINDINGS (OFFSPRING): Live birth index: 100% (test groups 0 and 1), 99% (test group 2) and 98 % (test group 3). 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.

Effect levels (F1)

Remarks on result:
other: Not determined

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion