Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 May to 05 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP
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)
Version / remarks:
adopted 22.03.96
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Batch number: 1K72J
Purity: 94.2%

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) 70 days
- Weight at study initiation: (P) Males: 339-399 g; Females: 225-277 g
- Housing: Animals were housed inside a barriered rodent facility.
- Diet (e.g. ad libitum): The animals were allowed free access to a standard rodent diet except overnight before routine blood sampling.
- Water (e.g. ad libitum): Potable water taken from the public supply was freely available via polycarbonate or polypropylene bottles fitted with sipper tubes.
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Photoperiod (hrs dark / hrs light): a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours

IN-LIFE DATES: Males From 2 July to 7 August 2012; Females From 2 July to 18 August 2012

No additional data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% Methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was prepared for administration as a series of graded concentrations in the vehicle. Starting with the lowest concentration, the required amount of the test substance was weighed, transferred to a suitably sized mortar and ground to a fine powder using a pestle. Small amounts of the vehicle were added and mixed with the test substance to form a smooth paste. The suspension was poured into a measuring cylinder which had been wetted with the vehicle. The mortar was rinsed thoroughly with the vehicle and the rinsed residue was added to the measuring cylinder. The required volume was achieved by addition of the remaining vehicle and the suspension was transferred to a beaker and mixed using a high shear homogeniser until homogenous. During magnetic stirring, the suspension was transferred to the final containers, via syringe. Remaining concentrations were prepared in ascending group order using the same method.

VEHICLE
- Concentration in vehicle: 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

No additional data
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: The day on which evidence of mating was found (at least one copulation plug or a sperm positive vaginal smear) was designated Day 0 of gestation.
- After successful mating each pregnant female was caged (how): Once mating occurred, the males and females were separated and smearing was discontinued.

No additional data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sample process
A representative sample of test formulation (nominally 1 mL, accurately weighed) was dissolved using ultrasonic vibration in a suitable volume of diluent (acetonitrile / water (80/20 v/v)). The extract was diluted using mobile phase to provide a solution containing the test substance at an expected concentration within the range 1.0 μg/mL to 2.0 μg/mL.
The concentration of the test substance in the final solution was quantified by high performance liquid chromatography using UV detection as detailed in the chromatographic section.

Typical chromatographic conditions
Analytical column: Agilent Poroshell 120EC C18, 2.7 μm, 100 × 4.6 mm
Column temperature: 40°C
Mobile phase: Acetonitrile/water/acetic acid 45/55/0.3 v/v/v
Flow rate: 1.00 mL/minute
Detector wavelength: UV, 235 nm
Injection volume: 5 μL
Run time: 5 minutes
Approximate retention time: 3.8 minutes

The mean concentrations of the test substance in test formulations analysed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Males were treated daily, two weeks before pairing up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing and gestation, until Day 6 of lactation.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 or 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
None stated
Positive control:
None stated

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each male was recorded before dose administration, on the day that treatment commenced (Week 0), weekly throughout the treatment period and before necropsy. Females were weighed before dose administration, on the day that treatment commenced (Week 0), weekly until mating was detected, on Days 0, 6, 13 and 20 after mating and on Days 1, 4 and 7 of lactation.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OTHER: Detailed physical examination and arena observations, Sensory reactivity and grip strength, Motor activity, Mortality, Haematology, peripheral blood, Blood chemistry, Oestrous cycles, Mating, Parturition observations and gestation length, Records made during littering phase, Necropsy and histology

No additional data
Oestrous cyclicity (parental animals):
For 15 days before pairing, daily vaginal smears (dry) were taken from all females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the males, smearing was continued using pipette lavage, until evidence of mating was observed.
Sperm parameters (parental animals):
None stated
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: Clinical signs, Litter size, Sex ratio, Bodyweight

No additional data
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Surviving males were killed in Week 6 after completion of the Week 5 investigations.
- Maternal animals: Females and offspring were killed on Day 7 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of: All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Many tissues were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of: For offspring surviving to scheduled termination, a careful external examination was performed for gross abnormalities and externally normal offspring were discarded without internal examination. Externally abnormal offspring were internally examined and any abnormal tissues were retained in an appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGTHS
Many tissues were prepared for microscopic examination.

No additional data
Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including oestrous cycles, pre-coital interval, mating performance gestation length and gestation index, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food consumption was analysed on a cage basis except before pairing. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Reproductive indices:
None stated
Offspring viability indices:
None stated

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male receiving 100 mg/kg/day was found dead on Day 28 of treatment. There were no in-life findings prior to death. This death was considered to be secondary to mis-dosing, and was not considered to be related to treatment with the test substance.
There were no signs recorded at routine observations or in association with dosing.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no dosage related effects of treatment upon male or female bodyweight.
Mean bodyweight and bodyweight gain was slightly higher for males receiving 300 mg/kg/day when compared with Controls. Overall bodyweight gain was slightly, but not statistically significantly higher for males receiving the test substance when compared with Controls, but no dose response was apparent.
Food consumption was considered not to be affected by treatment with the test substance up to 1000 mg/kg/day by males and females prior to pairing, during gestation and lactation when compared with Controls.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Assessment of oestrous cycles during the two week pre-pairing period showed that the majority of main phase females had regular 4/5 day oestrous cycles and it was considered that this parameter was not affected by treatment with the test substance.
One Control female was acyclic (at least ten days without oestrus).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ weights were not obviously affected by treatment with the test substance, when compared with Controls.
Mean adjusted spleen weights were statistically significantly higher in males receiving 1000 mg/kg/day, when compared with Controls; this was not evident in the females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The macroscopic examination of males after five weeks of treatment or on Day 7 of lactation revealed no intergroup differences.
The nature and incidence of all findings were consistent with the commonly seen background of macroscopic changes.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The following examined tissues had no findings microscopically: Adrenals, Brain, Caecum, Colon, Duodenum, Epididymides, Eyes, Ileum, Jejunum, LN Mesenteric, Liver, Lt. LN Axillary, Mammary, Peyer's Patch, Prostate, Sciatic Nerve, Seminal Vesicles, Skeletal Muscle, Skin, Spinal C. Cerv., Spleen, Sternum + Marrow, Stomach, Thymus, Thyroids, Trachea, Urinary Bladder

No additional data

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for general toxicity

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 specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
All females in all groups gave birth and raised their offspring successfully to Day 7 of age. The mean number of implantations, the live litter size on Day 1, offspring survival up to Day 7 of age and sex ratio were not affected by parental treatment.
Group mean post implantation survival index, mean live birth index, viability index and lactation index were unaffected by treatment with the test substance.

CLINICAL SIGNS (OFFSPRING)
Clinical signs recorded for the offspring identified occasional pups with findings, but the type of findings and incidence were typical of post-natal offspring (e.g. bruising) and showed no relationship to treatment.

BODY WEIGHT (OFFSPRING)
Mean offspring bodyweight on Day 1 of age and subsequent mean bodyweight gain up to Day 7 of age for males and females from animals treated at 100, 300 or 1000 mg/kg/day was not affected by treatment when compared with Controls.

GROSS PATHOLOGY (OFFSPRING)
There were no findings for offspring dying before scheduled termination that were considered related to treatment with the test substance.
There were no macropathology findings among offspring examined on Day 7 considered to be related to treatment.

No additional data

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
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: for reproductive /developmental toxicity

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
It is concluded that oral administration of the test substance to Crl: CD (SD) rats for at least 5 weeks at doses of 100, 300 and 1000 mg/kg/day was well tolerated with no toxicologically significant systemic effects and there was no effect of treatment on reproductive performance, including mating performance, fertility and offspring survival and development up to Day 7 of age.
For general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day and for reproductive /developmental toxicity the no-observed-adverse-effect-level (NOAEL) was also considered to be 1000 mg/kg/day.
Executive summary:

The influence of BMI on fertility and reproductive toxicity were tested in a combined repeat oral dose/reproductive toxicity screening test. The study was carried out in accordance with GLP and to international guidelines (OECD TG 422). Three groups, each comprising ten males and ten females received BMI at doses of 100, 300 or 1000 mg/kg/day. Males were treated daily, two weeks before pairing up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing and gestation until Day 6 of lactation. Bodyweight gain or food consumption (prior to pairing, gestating or lactating), oestrous cycles, pre-coital interval, mating performance and fertility or gestation length were not affected by treatment with BMI. For reproductive toxicity the NOAEL level was considered to be 1000 mg/kg/day.