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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: combined repeated dose and reproduction/developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-22 to 2013-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP and 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, OPPTS 870.3650 (Combined Repeated dose toxicity study with the reproduction/developmental toxicity screening test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid, pasty/colorless to yellow
Details on test material:
Please refer to confidential details on test material

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 old
- Housing: 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). For motor activity (MA) measurements the animals were housed individually in polycarbonate cages with small amounts of bedding material. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Air changes: 15 air changes per hour
- Photoperiod: The day/night cycle was 12 hours.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently released for 30-40 minutes in an ultrasonic bath. The test substance preparations were produced at least once a week. After the reduction of the dose level of the female animals from 40 mg/kg bw/d to 20 mg/kg bw/d the test substance preparations were produced for the first study week as follows:
30 mL of the high dose preparation (40 mg/kg bw/d) was filled up with 30 mL of corn oil to obtain the desired dose of 20 mg/kg bw/d.

VEHICLE
- Justification for use and choice of vehicle:
Corn oil is a standard vehicle for studies of this type.
- Concentration in vehicle:
0.625, 2.5, 5, 10 mg/L
Details on mating procedure:
MATING OF F0 GENERATION PARENTAL ANIMALS
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. 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 stability of the test substance in corn oil for a period of 7 days at room temperature was proven before the start of the study. Concentration control analysis of the test substance preparations were performed in samples of all concentrations at the start of the administration period. Given that the test substance is completely miscible with corn oil, solutions were considered to be homogenous without further analysis.
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 and two weeks thereafter in females.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0,2.5, 10 (both sexes), 20 [40 on pre-mating day 0] mg/kg bw/d (females only) and 40 mg/kg bw/d (male only).
Basis:

No. of animals per sex per dose:
10 male and 10 female animals
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Parental animals: Observations and examinations:
MORTALITY: 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.


CLINICAL OBSERVATIONS: Yes
- Time schedule: 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. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The following parameters were examined:

1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: 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 following exceptions are notable for the female animals:
1. 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.
2. Females with litter were weighed on the day of parturition (PND 1) and on PND 4.
3. 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.

FOOD CONSUMPTION: Yes
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
1. Food consumption was not determined during the mating period (male and female F0 animals).
2. Food consumption of the F0 females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
3. Food consumption of F0 females, which gave birth to a litter was determined for PND 1 - 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) and in males after the premating period.

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

HEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The following parameters were determined in blood:
- Leukocyte count (WBC),
- Erythrocyte count (RBC),
- Hemoglobin (HGB),
- Hematocrit (HCT),
- Mean corpuscular volume (MCV),
- Mean corpuscular hemoglobin (MCH),
- Mean corpuscular hemoglobin concentration (MCHC),
- Platelet count (PLT),
- Differential blood count,
- Reticulocytes (RET),
- Prothrombin time (Hepato Quick’s test) (HQT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: please refer to hematology
The following clinicochemical parameters were determined:
- Alanine aminotransferase (ALT),
- Aspartate aminotransferase (AST),
- Alkaline phosphatase (ALP),
- γ-Glutamyltransferase (GGT),
- Sodium (NA),
- Potassium (K),
- Chloride (CL),
- Inorganic phosphate (INP),
- Calcium (CA),
- Urea (UREA),
- Creatinine (CREA),
- Glucose (GLUC),
- Total bilirubin (TBIL),
- Albumin (ALB),
- Globulins (GLOB),
- Triglycerides (TRIG),
- Cholesterol (CHOL),
- Bile acids (TBA).

URINALYSIS: Yes
- Time schedule for collection of urine: For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence.
The following parameters were determined:
- pH,
- Protein,
- Glucose,
- Ketones,
- Urobilinogen,
- Bilirubin,
- Blood,
- Specific gravity,
- Sediment,
- Color, turbidity,
- Volume.

NEUROBEHAVIOURAL EXAMINATION: Yes
1. Functional observation battery
A functional observational battery was performed in the first five male animals per test group and the first 5 female animals with litter of all test groups at the end of the administration period. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests.

Home cage observation:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Impairment of gait

Open field observation:
1. Behavior when removed from cage
2. Fur
3. Skin
4. Salivation
5. Nose discharge
6. Lacrimation
7. Eyes/ pupilsize
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Impairment of gait
15. Activity/ arousal level
16. Feces excreted within 2 minutes (number/ appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 minutes

Sensory motor tests/reflexes:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Auditory startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

2. Motor activity assessment
Motor activity (MA) was also measured on the same day as the FOB was performed in the first five parental males and females (with litter) per group.
Litter observations:
Litter/Pups
- Pup number and status at delivery
- Sex ratio
- Pup clinical observations
- Pup body weight data
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 was given to the reproductive organs.

ORGAN WEIGHTS
The following weights were determined in all males sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Testes
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

HISTOPATHOLOGY
The following organs were examined:
- Adrenal glands,
- All gross lesions,
- Bone marrow (femur),
- Brain,
- Cecum,
- Cervix,
- Coagulating glands,
- Colon,
- Duodenum,
- Epididymides,
- Heart,
- Ileum,
- Jejunum,
- Kidneys,
- Liver,
- Lungs,
- Lymph nodes (axillary and mesenteric),
- Ovaries,
- Oviducts,
- Prostate gland,
- Peyer’s patches,
- Rectum,
- Sciatic nerve,
- Seminal vesicles,
- Spinal cord (cervical, thoracic, lumbar),
- Spleen,
- Stomach (forestomach and glandular stomach),
- Testes,
- Thymus,
- Thyroid glands,
- Trachea,
- Urinary bladder,
- Uterus,
- Vagina.
Postmortem examinations (offspring):
- Pup necropsy observations
Statistics:
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, implantation sites, pups delivered, life pups day:
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 delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups:
Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions.

Mating days until day 0 pc:
Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians.

Viability index:
Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians.

Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the hypothesis of equal medians.

Statistics of clinical pathology and pathology:
Please refer to any other information on material and methods incl. tables
Reproductive indices:
Male reproduction data:
- male mating index (%)
- male fertility index (%)

Female reproduction and delivery data:
- female mating index (%)
- female fertility index (%)
- gestation index (%)
- live birth index (%)
Offspring viability indices:
- Pup viability/mortality (Viability index (%))

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

MORTALITY
Three female animals of the high dose group were found dead when treated with 40 mg/kg bw/d on premating study day 1. Following reduction of the dose level for high dose group females from 40 to 20 mg/kg bw/day, there were no further incidences of mortality.

DETAILED CLINICAL OBSERVATION

Summary of clinical obserevations for males and females:
- Nine female animals of the 40 mg/kg bw/d dose group showed severe tremors on premating day 1. Three females of the 40 mg/kg bw/d dose group were found dead on premating day 1. In response, the dose level was reduced to 20 mg/kg bw/d in female animals for the remainder of the treatment period.
- Three male animals (40 mg/kg bw/d), one female animal (20 [40 on pre-mating day 0] mg/kg bw/d) and two female animals (10 mg/kg bw/d) showed salivation during the premating period.
- One male animal (2.5 mg/kg bw/d), four male animals (40 mg/kg bw/d) and one female animal (10 mg/kg bw/d) showed salivation during the mating period.
- Two male animals (40 mg/kg bw/d) showed salivation during the postmating period.

The salivation in different test groups on different time points in both sexes was assessed as substance-related but not as an adverse effect, due to the taste of the substance.

Summary of clinical obserevations for females during gestation:
One female animal (10 mg/kg bw/d) showed slight salivation. This finding was assessed as substance-related but not as an adverse effect, due to the taste of the substance.
Clinical observation for females during lactation:
No clinical signs were observed.

BODY WEIGHT DATA
No deviations in the body weights of all test groups were observed.

FOOD CONSUMPTION
Female animals (10 mg/kg bw/d) showed increased food consumption values on gestation day 7 (+8.2%) compared to the control group.
No other significant deviations in male and female animals were observed during the study period; therefore no dose response relationship was observed and this finding was assessed as being incidental.

WATER CONSUMPTION
No test substance-related findings were noted.

HEMATOLOGY
No treatment-related changes among hematological parameters were observed. In males of the 2.5 and 10 mg/kg bw/d dose group mean corpuscular volume (MCV) was increased and additionally in males of the 2.5 mg/kg bw/d dose group, mean corpuscular hemoglobin content (MCH) was higher compared to controls. Both calculated parameters were not dose-dependently changed and the measured red blood cell parameters (i.e., red blood cell (RBC) counts, hemoglobin concentration and hematocrit) were not altered in these animals. Therefore, these changes were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
- Home cage observations: No test substance-related effects were observed.
- Open field observations: No test substance-related effects were observed.
- Sensorimotor rests/reflexes: No test substance-related effects were observed.
- Quantitive parameters: No test substance-related effects were observed.
- Motor activity measurement: There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals.

ORGAN WEIGHTS
When compared to the control group (set to 100%), the mean absolute liver weights were significantly increased in male animals of the 2.5 mg/kg bw/d dose group and of the 40 mg/kg bw/d dose group. All other mean absolute weight parameters in males and all weight parameters in females did not show significant differences when compared to the control group. None of the mean relative weight parameters showed significant differences when compared to the control group. Absolute liver weights of male animals did not show a dose response, there were no significant changes in relative liver weights, and a histopathological correlate was not detected, therefore increased absolute liver weights were regarded as incidental.

GROSS LESIONS
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment. None of the animals premature decents showed any gross lesions.

GROSS LESIONS - FERTILITY
Two mating pairs of the 2.5 mg/kg bw/d dose group, one pair of the 10 mg/kg bw/d dose group and three pairs of the 40/20 mg/kg bw/d dose group were recorded as no offspring/ not pregnant.
Mating pairs of the 2.5 mg/kg bw/d dose group and of the 10 mg/kg bw/d dose group did not show gross lesions. Two male animals of the 40 mg/kg bw/d dose group showed relevant macroscopic lesions: A focus was observed on the right epididymis of a male animal, and the organ size of the left epididymidis was reduced in other male animal.

HISTOPATHOLOGY
All findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. There were no relevant histopathological findings in any of the animals having died spontaneously.
HISTOPATHOLOGY - FERTILITY
Two mating pairs of the 2.5 mg/kg bw/d dose group, one pair of the 10 mg/kg bw/d dose group and three pairs of the 40/20 mg/kg bw/d dose group were recorded as no offspring/ not pregnant.
Among these mating pairs, two male animals (Nos. 32 and 39 ) of the 40 mg/kg bw/d dose group both showed a unilateral sperm granuloma in the epididymis correlating to “focus” macroscopically in animal No. 32. Male animal No. 39 showed additionally immature (not dilated, empty) ducts of the contralateral epididymis, correlating to reduced size of this organ at necropsy. The testis of that side was missing in animal No. 39.

MALE REPRODUCTION DATA
- Male mating index: The male mating index was 100% in all test groups.
- Male fertility index: Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. One male of the 10 mg/kg bw/d dose group and 2 males of the 2.5 mg/kg bw/d dose group did not generate F1 pups. The male fertility index was 80% in the 2.5 mg/kg bw/d dose group, 90% in the 10 mg/kg bw/d dose group and 100% in the 40 mg/kg bw/d dose group and the control group. 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.

FEMALE REPRODUCTION AND DELIVERY DATA
- Female mating index: The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 3.1, 2.5, 1.8 and 1.6 days in the control, low, mid and high dose groups, respectively. 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.
- Female fertility index: The female fertility index was 80% in the 2.5 mg/kg bw/d, 90% in the 10 mg/kg bw/d and 100% in the 20 [40 on pre-mating day 0] mg/kg bw/d dose group and the control group. Female animals , which delivered no pups, showed no implantation sites. The mean duration of gestation was similar in all test groups. 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.
- Gestation index: The gestation index was 100% in all test groups. Live birth indices: The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 99% - 100% in all test groups.
- Postimplantation loss: The postimplantation loss was 3.38 % in the control group, 0.96 % in the 2.5 mg/kg bw/d dose group, 6.62 % in the 10 mg/kg bw/d dose group and 1.02 % in the 20 [40 on pre-mating day 0] mg/kg bw/d dose group. 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.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: There were no signs of systemic toxicity at the highest dose level of 40 mg/kg bw/day in male parental animals.
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
(reproductive performance and fertility)
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No reproduction effects were noted up to the highest dose tested in male animals.
Dose descriptor:
NOAEL
Remarks:
(reproductive performance and fertility)
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No reproduction effects were noted up to the highest dose tested in female animals.

Results: F1 generation

Details on results (F1)

F1 GENERATION LITTER/PUPS

- Pup number and status at delivery:The mean number of delivered F1 pups per dam was evenly distributed among the groups.

- Pup viability/mortality: The viability index indicating pup mortality during lactation (PND 0 - 4) was 98.7% (20 [40 on pre-mating day 0] mg/kg bw/d dose group), 99.1% (10 mg/kg bw/d dose group) 100% (2.5 mg/kg bw/d dose group and control group) and was in the normal range of biological variation inherent in the strain of rats used for this study. One female pup of control group and 10 mg/kg bw/d dose group were found stillborn. 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.
- Sex ratio: 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.
- Pup clinical observations: The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
- Pup body weight data: Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group. Four male runts were seen in the 2.5 mg/kg bw/d dose group. 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.
- Pup necropsy observation: One F1 pup of the 2.5 mg/kg bw/d dose group showed post mortem autolysis and one pup of the 20 mg/kg bw/d dose group was cannibalized. These findings were assessed as being spontaneous in nature and without biological relevance.

Effect levels (F1)

Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance-related, advers findings were observed
Remarks on result:
other: highest tested dose

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

DOSE FORMULATION ANALYSIS

The stability of the test substance in corn oil was demonstrated over a period of 7 days at room temperature. The analytically determined test substance concentrations in the vehicle were in the expected range of the nominal concentrations (91.5 -103.2%) for all formulations.

Applicant's summary and conclusion