Registration Dossier

Administrative data

Description of key information

A preliminary 7-day repeat oral dose study was carried out in accordance with GLP (Huntingdon Life Sciences, 2012) in order to select a suitable high dose for a subsequent study. Groups of rats were dosed at 100, 300 or 1000 mg/kg/day. There was no treatment related toxicity, therefore the limit dose level of 1000 mg/kg/day was considered acceptable for use as the notional limit high dose in the subsequent screening study.

A combined repeat oral dose/reproductive toxicity study was carried out in accordance with GLP and to international guidelines (Huntingdon Life Sciences, 2012, study LBA0061). Groups were dosed at 100, 300 or 1000 mg/kg/day. No significant adverse effects were observed, therefore the NOAEL was considered to be 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
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: The study was run according to OECD test guidelines and in compliance with GLP; on thia basis it is considered reliable without restrictions.
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)
Limit test:
no
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: From: 2 July 2012 To: Males 7 August 2012, Females 18 August 2012

No additional data
Route of administration:
oral: gavage
Vehicle:
other: 1% Methylcellulose
Details on oral 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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Apparatus and instrumentation
High performance liquid chromatograph (HPLC)

Sample process
A representative sample of test formulation (nominally 1 mL, accurately weighed) was dissolved using ultrasonic vibration in a suitable volume of diluent. The extract was diluted using mobile phase to provide a solution containing 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.
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
Observations and examinations performed and frequency:
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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 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

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 2 (prior to pairing)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: the first five surviving males and females in each group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 2 (prior to pairing)
- Animals fasted: Yes
- How many animals: the first five surviving males and females in each group

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

No additional data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, 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: Yes
Other examinations:
None stated
Statistics:
None stated
Clinical signs:
no effects observed
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
One male receiving 100 mg/kg/day was found dead on Day 28 of treatment. There were no in-life findings prior to this animals death. Post mortem examination revealed a perforated oesophagus; the thorax contained clear fluid with pale adhesions, dark lungs and bronchi and reduced contents of the colon, ileum and caecum. This death was not considered related to treatment.
There were no signs recorded at routine observations or in association with dosing.

BODY WEIGHT AND WEIGHT GAIN
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.
During the pre-pairing phase mean bodyweight gain for females receiving the test substance up to 1000 mg/kg/day was slightly lower than that of the Control values, however, there was no dose response. Mean bodyweight and bodyweight gain was slightly higher during the gestation phase, but slightly lower than Controls during the lactation phase for females receiving the test substance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
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.
Group mean food intake was statistically significantly higher during Days 0-5 of gestation for females receiving 300 or 1000 mg/kg/day; however, this slight increase was of no biological importance.

HAEMATOLOGY
There were no consistent findings at haematological investigations to suggest any adverse dose response to treatment with the test substance. Haematological investigation in Week 2 of treatment revealed, occasional parameters which were statistically significantly different from Control but there was no clear biological significance of dosage relationship.

CLINICAL CHEMISTRY
The biochemical examination of the blood plasma in Week 2 of treatment detected no consistent changes suggestive of treatment related effect. Sporadic intergroup differences which appeared statistically significant included increased urea, creatinine and potassium levels in males and increased glucose levels in animals receiving 100 mg/kg/day when compared with Controls; in the absence of similar findings at higher doses a relationship to treatment is uncertain. Cholesterol and bile acids were lower in males receiving 1000 mg/kg/day when compared with Controls; there was no dose response for the cholesterol data and the bile acid data was highly variable.

ORGAN WEIGHTS
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
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: NON-NEOPLASTIC
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
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:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Critical effects observed:
not specified
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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A preliminary 7-day oral toxicity study was conducted (Huntingdon Life Sciences, 2012) to assess the systemic toxic potential of BMI and to select a suitable high dose for a subsequent OECD 422 main study. The study was conducted in accordance with OECD and EC test guidelines and in compliance with GLP. Three groups, each comprising three male and three female rats received BMI at doses of 100, 300 or 1000 mg/kg/day. There were no premature deaths, no effect on bodyweight, no effect on food consumption, organ weights and no signs at necropsy indicative of a reaction to treatment. In the absence of any treatment related toxicity in this study the limit dose level of 1000 mg/kg/day was considered acceptable for use as the notional limit high dose in the subsequent OECD 422 screening studies.

A combined repeat dose toxicity and reproductive/developmental toxicity screening study in Rat was conducted (Huntingdon Life Sciences, 2012). The study was conducted in accordance with OECD and EU test guidelines and in complicance with GLP. Three groups, each comprising ten males and ten females received BMI at doses of 100, 300 or 1000 mg/kg/day. Males were dosed for up to five consecutive weeks. No treatment related adverse effects were noted in the study, therefore for general toxicity the NOAEL was considered to be 1000 mg/kg/day.

No repeat dose inhalation or dermal studies were undertaken as data from a reliable repeat dose oral study was already available and so inhalation and dermal studies were not required.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Study carried out as a combined repeat dose oral toxicity and repro tox. study according to GLP and to international guidelines. Study is thus reliable and has been designated the key study.

Justification for classification or non-classification

The NOAEL for the rat combined repeat dose toxicity and reproductive/developmental toxicity screening study in Rat was 1000 mg/kg/day. On this basis BMI is not classified as toxic to health by prolonged exposure by the oral route.