Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 December 2018 (Study Initiation) to 9 May 2019 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

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
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals ; For mating, each female rat was placed with a male rat from the same dose level (1:1 mating) until mating occured or 2 weeks had elapsed.
- Basis for dose level selection ; Dose levels were selected based on the results of the dose range finding (DRF) study (JRF Study N° 410-1-02-21402). In the DRF study, no treatment related effects were observed on body weight, body weight gain, Food consumption and organ weight (absolute and relative) up to 1000 mg/kg b. wt./day. Therefore, the following dose levels, 100, 300 and 1000 mg/kg b. wt./day, were selected for the main OECD 422 study.
- Inclusion/exclusion of extension of Cohort 1B; n/a
- Termination time for F2 ; n/a
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B ; n/a
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 ; n/a
- Route of administration ; oral by gavage
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable];
The preferred species is the rat as it has been historically shown to be a suitable model for developmental and reproductive toxicity testing and is recommended by the OECD, EC and other regulatory authorities. In this study, healthy adult rats (Rattus norvegicus) of Wistar strain (RccHan:WIST), an accepted test strain, were used.
Based on solubility performed at JRF (JRF Study N° 410-1-02-21402) and after consultation with the Sponsor, 0.5% carboxy-methyl-cellulose (CMC) with 1% tween 80 was selected as the vehicle. 70 male and 70 female rats were selected for testing, rats were divided into four main groups with an additional two recovery groups (control and high dose).The main groups had 15 rats/sex/group whereas recovery groups had 5 rats/sex/group.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities Pvt Ltd
- Lot/batch No.of test material: MNST9H796L
- Expiration date of the lot/batch: August 2023
- Purity test (release): September 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), in original container as supplied by the Sponsor. Container kept tightly closed in a dry, cool and well ventilated place
- Stability under test conditions: The stability of the active ingredient (a.i.) concentration in the vehicle was determined prior to initiation of the study using a validated analytical method (JRF Study N° 228-2-13-21401). Based on the results obtained, the test item in the vehicle was stable for 4 days.
- Solubility and stability of the test substance in the solvent/vehicle: Assumed stable for the duration of the study when dose formulations were prepared within stability parameters. The control and high dose groups were prepared daily, and dose formulations of low and mid dose groups were prepared a minimum of thrice weekly.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, test item prepared in the vehicle and mixed using a magnetic stirrer

FORM AS APPLIED IN THE TEST (if different from that of starting material) None, test item prepared in the vehicle and mixed using a magnetic stirrer

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Not specified

Test animals

Species:
rat
Strain:
Wistar
Remarks:
(RccHan:WIST)
Details on species / strain selection:
The preferred species is the rat. It has been historically shown to be a suitable model for developmental and reproductive toxicity testing and is recommended by the regulatory authorities. The results may be of value in predicting the toxicity of the test item to humans.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Healthy adult rats (Rattus norvegicus) of Wistar strain (RccHan:WIST) were obtained from the Animal Breeding Facility, JRF.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 11-12 wks; (F1) 11-12 wks
- Weight at study initiation:
(P) Males: Group 1 (364.0-442.0g), Group 2 (354.8-452.5g), Group 3 (362.6-438.0g), Group 4 (363.5-427.8g), Rec Group 5 (377.1-433.2g). Rec Group 6 (373.2-414.2g)
(P) Females: Group 1 (236.9-294.4g), Group 2 (236.9-296.9g), Group 3 (241.3-292.7g), Group 4 (241.6-310.6g), Rec Group 5 (243.9-368.3g), Rec Group 6 (233.4-279.8g)
- Fasting period before study: no
- Housing: Throughout the experiment period, the male and female rats were housed in groups of 2 or 3 rats/sex/cage. Except, during the mating period, rats were housed in groups of 2 rats/cage (one male plus one female) and mated female rat was caged individually. Enrichment material was provided to all animals. Nesting material was provided at near parturition (from gestation day 17). During study, rats were housed in solid floor polypropylene rat cages (size: 41 cm x 28.2 cm x 18 cm). Each cage were fitted with a stainless steel top grille having provision for polypropylene water bottle with stainless steel drinking nozzle. The bottom of cages were layered with clean sterilised rice (paddy) husk as bedding material. Cages were placed on 5 tier racks. Cages and enrichment material were changed a minimum of twice a week. Cages were arranged in such a way that any possible effects due to cage placement were minimised.

- Diet: Rats were fed ad libitum with standard rodent diet (Teklad Certified Global 16% Protein Rodent Maintenance Diet, Batch N° 2016SC-050818MA from Envigo Laboratories, Inc., USA).
- Water: Rats were provided reverse osmosis (RO) water ad libitum (from a RO water filtration system) in polypropylene bottles.
- Acclimation period: 5 days before initiation of pre-treatment estrous cycle evaluation
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C
- Humidity (%): 57 - 66%
- Air changes (per hr): ≥15
- Photoperiod (hrs dark / hrs light): The photoperiod was 12 hours light and 12 hours darkness, fluorescence light hours being 06.00 - 18.00 hours.
IN-LIFE DATES: From: To: 8 December 2018 (Experimental start) to 9 May 2019 (Experimental completion)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxy-methyl-cellulose (CMC) with 1% tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of the test item was received from JRF’s Test Item Control Office. The test item was mixed with 1% tween 80 (i.e. 1% of total volume to be prepared) followed by mixing with 0.5% CMC to make the dose formulations of required concentrations. The prepared dose formulations were thoroughly mixed using a magnetic stirrer before dosing.

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on a solubility study performed at JRF (JRF Study N° 410-1-02-21402), 0.5% carboxy-methyl-cellulose (CMC) with 1% tween 80 was selected as the vehicle.
- Concentration in vehicle: 0 (vehicle), 10 mg/kg (low dose), 30 mg/kg (mid dose) and 100 mg/kg (high dose), dosed at a dose volume of 10 mL/kg bw.
- Amount of vehicle (if gavage): The test item was mixed with 1% tween 80 (i.e., 1% of total volume to be prepared) followed by mixing with 0.5% CMC to make the dose formulations of required concentrations.

Name Batch / Lot N° Manufactured by
Carboxy methyl cellulose QF5Q651243 Merck Life Science, Pvt. Ltd.
Tween 80 #BCBW9985 Sigma Aldrich

Details on mating procedure:
- M/F ratio per cage: During the mating period, male and female rats were mated in a 1:1 ratio to obtain the F1 pups.
- Length of cohabitation: For mating, each female rat was placed with a male rat from the same dose level (1:1 mating) until mating occured or 2 weeks had elapsed.
- Proof of pregnancy: Day 0’ of pregnancy is defined as the day on which sperm is observed in the vaginal smear of rats.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. no
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: mated female caged individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and Active Ingredient Concentration of the Test Item in the Vehicle
The active ingredient (a.i.) concentration and homogeneity of dose formulations were analysed by the Department of Chemistry, using the validated analytical method (JRF study N° 228-2-13-21401). The mean percent recovery obtained for the test doses was within the acceptance level of ±15% of the nominal concentration. This demonstrated that the exposure concentrations were as intended with a %CV of less than 10, confirming test doses were homogeneously prepared.
Duration of treatment / exposure:
Male: two weeks prior to mating, during the mating (two weeks) and until approximately 80% of the females had delivered. Total No. of treatment days was 47.
Female: two weeks prior to mating, the variable time to conception, the duration of pregnancy and fourteen days after delivery.
Frequency of treatment:
The vehicle and dose formulations were administered to male and female rats daily up to and including the day before scheduled sacrifice.
Details on study schedule:
Only parental (P0) animals were mated.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
G1 - Control (0.5% carboxy-methyl-cellulose (CMC) with 1% tween 80)
Dose / conc.:
100 mg/kg bw/day
Remarks:
G2 - Low dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
G3 - Mid-dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
G4 - High dose
Dose / conc.:
0 mg/kg bw/day
Remarks:
G5 - Control (recovery group)
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
G6 - High dose (recovery group)
No. of animals per sex per dose:
Main groups: 15 rats/sex/group.
Recovery groups: 5 rats/sex/group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of the dose range finding (DRF) study (JRF Study N° 410-1-02-21402). In the DRF study, no treatment related effects were observed on body weight, body weight gain, food consumption and organ weight (absolute and relative) up to 1000 mg/kg b. wt./day. Therefore, the following dose levels, 100, 300 and 1000 mg/kg b. wt./day, were selected for the main OECD 422 study.
- Rationale for animal assignment (if not random): 70 male and 70 female rats were subjected to randomisation using an in-house developed and validated computer software program.
- Fasting period before blood sampling for clinical biochemistry: yes
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: yes
- Time schedule: Rats were observed twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: Rats were observed twice daily during treatment and recovery periods for any visible clinical signs.

BODY WEIGHT: yes
- Time schedule for examinations: Male: The body weight of all male rats was recorded on the first day of dosing and at weekly intervals thereafter. The body weight of all female rats was recorded on the first day of dosing and at weekly intervals during the pre-mating period.

During the gestation period, female rats were weighed on gestation day 0, 7, 14, and 20. During the lactation period, female animals were weighed within 24 hours of parturition (day ‘0’ post-partum/lactation day), and on post-partum days 4, 7, 14 and on the day of terminal sacrifice. Parturition day ‘0’ is defined as the day on which the female littered.
The body weight of all rats belonging to recovery groups was recorded on first day of dosing and at weekly intervals thereafter. On the day of fasting, body weights of all surviving rats were recorded. Body weights of all rats were also recorded on the day of sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food weights of male rats were determined weekly during the pre-mating and post-mating periods.In female rats, during the pre-mating period, food weights were recorded at weekly intervals.
During the gestation period, food weights were measured on days 0, 7, 14, and 20. During the lactation period, food weights were measured on days 0, 4, 7, and 14.
Food consumption was not measured during the mating period.
Food weights of male and female rats belonging to recovery groups were determined weekly throughout treatment and recovery periods.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
Oestrous cycle length and pattern were evaluated by vaginal smears of individual female rats during the two week pre-treatment period. Vaginal smear were monitored daily from the beginning of the treatment period until evidence of mating. A vaginal smear, from each pregnant animal, was also observed on the day of terminal sacrifice. Care was taken to avoid disturbance to mucosa while obtaining vaginal cells.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight,
Levator ani plus bulbocavernosus muscle complex (LABC), Cowper’s glands, Glans penis,
Thyroid gland, Seminal vesicles with Coagulating gland, Prostate (dorsolateral and ventral),
Gross lesions, male mammary gland, Pituitary

Organ Weights: Testes, epididymis, Levator ani plus bulbocavernosus muscle complex (LABC), Cowper’s glands, glans penis, prostate and seminal vesicles with coagulating glands as a whole, thyroid.

Detailed testicular histopathological examination (paraffin embedding and transverse sections of 4-5µm thickness) were conducted with special emphasis on the stages of spermatogenesis and the histopathology of interstitial testicular cell structure. The evaluation was to identify treatment related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis included the caput, corpus and cauda, and accomplished by evaluation of a longitudinal tissue section. The epididymis was evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types and phagocytosis of sperm. Periodic Acid Schiff (PAS), hematoxylin and eosin staining were used for examination of the testes, while hematoxylin and eosin was used for epididymides.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes , on postnatal day (PND) four, the size of each F1 litter was adjusted by removing extra pups by random selection to yield, as close as possible, four male and four female pups per litter. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (e.g., six males and two females) was performed. Adjustments were not performed for litters of eight pups or less.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Pups Observation: Each litter was examined as soon as possible after delivery to establish the number of pups, sex of pups, stillbirths, live birth, runts, and the presence of gross anomalies. Pups which died during lactation were weighed and subjected to post-mortem examination. Pups found dead on the day of littering were examined for possible defects and cause of death, these were discarded in the absence of any gross findings.
Each pup was observed for the presence of milk in the stomach on PND 0 to ensure adequate paternal the nursing care.
The AGD of each pup was measured on PND 0. Male pups were observed for the retention of nipples/areolae on PND 13.

GROSS EXAMINATION OF DEAD PUPS: yes, gross examination of rats, including pups was conducted.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Neurobehavioral Observation (NBO): To assess the behavioral and neurological status of each rat, the following parameters of NBO were evaluated prior to initiation of treatment and at weekly intervals, thereafter. IIn home cage, all rats from treatment and control groups revealed normal postures asleep (curled up often asleep), sitting A (sitting but with head hung down), sitting B (sitting normally, feet tucked in), sitting C (sitting or standing alert, watching) and rearing. Clonic and tonic movements were absent in home cage during NBO.

Handling Observations
NBO performed during handling of rats did not reveal any abnormality related to treatment. All the rats revealed normal behavior during removal (very easy - animals sit quietly) and handling (easy - alert, limbs put against the body). None of the rats showed lacrimation, salivation or piloerection. Eyelids were wide open in all rats. Eye and skin examination of rats from all groups did not reveal any abnormality.

Open Field Observations
In open field, all rats from treatment and control groups showed normal gait, mobility, arousal and respiration during the three minute observation period. Clonic and tonic movements, stereotypy and bizarre behavior were absent. No treatment related significant changes were observed in vocalisation, rearing, and urination and defecation counts of male and female rats from treatment groups when compared with that of the control group.
Some incidental changes in male rats were noted viz., statistically significant decrease in rearing count at week 2 in low and mid dose groups; urination count in mid dose group at week 5 when compared with that of the control group.
Some incidental changes in female rats were noted viz., statistically significant decrease in rear count at pre-treatment in mid dose group and at 7 week in high dose group when compared with that of the control group.
These changes were considered as incidental in nature based on absence of dose dependent effect.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving male rats were sacrificed after approximately 80% of female animals had delivered.
- Maternal animals: All surviving animals were sacrificed on LD 15. Female rats were sacrificed on Lactation day (LD) 15. Females which did not mate were sacrificed 25 days after the last day of the mating period. Females which had not did not delivered by day 25 post-coitum were sacrificed on post-coitum day 25. Rats belonging to recovery groups were sacrificed after 14 days after the first scheduled sacrifice of dams.

GROSS NECROPSY
- After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened, and a thorough examination of organs was carried out to detect abnormalities. Gross examination of rats, including pups was conducted.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of sacrifice, the following organs/tissues of rats were excised, weighed, and fixed in appropriate fixatives to permit microscopic examination:
All adult rats:
Testes
Epididymis
Levator ani plus bulbocavernosus muscle complex (LABC)
Cowper’s glands
Glans penis
Ovary
Thyroid gland
Seminal vesicles with Coagulating gland
Prostate (dorsolateral and ventral)
Uterus with oviducts and cervix
Gross lesions (preserved)
Vagina (preserved)
Male mammary gland (preserved)
Pituitary (preserved)
ular, mesenteric)

5 adult rats/sex/group:
Liver
Kidneys
Adrenals
Spleen
Brain (cerebrum, cerebellum, and medulla/pons)
Heart
Spinal cord (at three levels; cervical, mid-thoracic and lumbar) (Preserved)
Stomach (preserved)
Small intestine (Duodenum, jejunum, ileum) (Preserved)
Large intestine (Caecum, colon, rectum) (Preserved)
Eye (Preserved)
Trachea (Preserved)
Lungs (preserved by inflation with fixative and then immersion) (Preserved)
Urinary bladder (Preserved)
Lymph nodes (mandibular, mesenteric) (Preserved)
Sciatic nerve (Preserved)
Skeletal muscle and bone (Preserved)
Bone marrow (Femur) (Preserved)


Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were not selected as parental animals were sacrificed on PND 14.
- These animals were subjected to postmortem examinations macroscopic and/or microscopic examination. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

GROSS NECROPSY
Gross necropsy was conducted under direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, animals were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the animal. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened and a thorough examination of the organs was carried out to detect abnormalities. Gross examination of rats, including pups was conducted.

HISTOPATHOLOGY / ORGAN WEIGTHS
Detailed histopathological examination of preserved organs (except LABC and glans penis) including gross lesions was performed in high and control dose group animals.
Detailed testicular histopathological examination, paraffin embedding and transverse sections of 4-5 µm thickness) was conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation included identification of treatment related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis included the caput, corpus and cauda, which was accomplished by evaluation of a longitudinal section. The epididymis was evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types and phagocytosis of sperm. Periodic Acid Schiff (PAS) and, hematoxylin and eosin staining were used for examination of the testes, while hematoxylin and eosin was used for epididymides and ovaries.
Histopathological examination of the ovary was carried out to detect treatment related effects such as qualitative depletion/increase of primordial, secondary, antral, graffian follicles population, persistence and increased/decreased corpus luteum, ovarian degeneration/atrophy and stromal cell proliferation.
Additionally histopathological examination of Cowper’s gland from all main (G1 to G4) and recovery group (G5 and G6) animals was carried out to detect treatment related effect as significant increase was observed in absolute weight.

ORGAN WEIGHTS, Yes, at the time of sacrifice, the relavent organs/tissues were excised, weighed, and fixed in appropriate fixatives to permit microscopic examination:
Statistics:
Statistical analysis was carried out using validated software developed at JRF. Non-pregnant rats were excluded from statistical analysis. Assumed pregnant rats were excluded from statistical analysis of gestation phase parameters.

Data such as body weight, body weight gain, food consumption, hind limb foot splay, grip strength, organ weight, organ weight ratio, number of implantation, pre-natal loss, post-natal loss, male sex ratio, estrous cycle length, haematology and clinical chemistry parameters, some urinalysis parameters and litter parameters (pups body weight and pups body weight gain) were subjected to Shapiro-Wilk’s test for checking normality wherever applicable, followed by Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data did not meet the normality, data was transferred to log form to check the normality again. Where log transfer data did not meet the normality, a Kruskal-wallis test was performed to calculate significance. Where the data did not meet the homogeneity of variance, statistical analysis was extended following a decision tree as provided by Gad, S.C., 2007. AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.

Count data {viz., litter size, number of implants, pre-coital interval, duration of gestation, number of estrous cycles, urination count, defecation count, rearing count, motor activity (total, fine and ambulatory)} were subjected to non-parametric test, either the Kruskal-wallis test or Mann-whitney test.

Non-parametric data such as gestation index, parturition index, pregnancy rate, survival index, mortality index, live birth index and fertility index were analysed using a Chi-Square test.

Flags for any significant difference between control and treated groups are:
Single arrow for p≤0.05
Double arrows for p≤0.01)

Reproductive indices:
No data
Offspring viability indices:
No data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Treatment related decrease in number of implants and number of pups counts at 300 and 1000 mg/kg b. wt./day.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
other: Post-natal growth

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
uterus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Effect levels (P1)

Remarks on result:
not measured/tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase in pup mortality was observed during PND 0 – 4 in male, female and composite of male and female pups belonging to mid and high dose (except male pup) groups when compared with that of the control group. Statistically significant increase in pup mortality was observed during PND 7-14 in composite of male and female pups belonging to low dose group when compard with that of the control group. These changes observed were considered as unrelated to treatment in the absence of any dose reponse trend.
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Statistically significant increase in body weight in female pups and composite of male and female pups on PND 4 and PND 4R (only in female pups) was observed in high dose group when compared with that of the control group. Statistically significant increase in body weight gain during PND 4R - 7 in male, and composite of male and female pups was observed in high dose group when compared with that of the control group. Increase in body weight is toxicologically insignificant, hence considered as unrelated to treatment.
Food efficiency:
not examined
Description (incidence and severity):
Statistically significant increase in body weight in female pups and composite of male and female pups on PND 4 and PND 4R (only in female pups) was observed in high dose group when compared with that of the control group. Statistically significant increase in body weight gain during PND 4R - 7 in male, and composite of male and female pups was observed in high dose group when compared with that of the control group. The increase in body weight is toxicologically insignificant and therfore considered as non-treatment related.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
no
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
It was concluded that:

- No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 1000 mg/kg b. wt./day based on no effect on systemic end-points.
- NOAEL for fertility toxicity was 1000 mg/kg b. wt./day based on no effect on fertility.
- The NOAEL for reproductive toxicity was 100 mg/kg b. wt./day based on treatment related decrease in the number of implants and pup count at 300 and 1000 mg/kg b. wt./day.
- The NOAEL for developmental toxicity was 1000 mg/kg b. wt./day dose level based on no effect on mortality, clinical sign, and, postnatal growth.