Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26 March to 23 April 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 96.5%
- Lot/batch No.: 5ABK-003

Test animals

Species:
rat
Strain:
other: Crl:CD®BR VAF PLUSTM
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Age at study initiation: 42±1 days old
- Weight at study initiation: A weight range of 74-85 g for males and 70-80 g for females
- Fasting period before study:
- Housing: All remaining rats were initially caged, as far as possible, in groups of five according to sex in metal cages with wire mesh floors. Each cage measured 35.8 cm wide, 53 cm deep and 25.7 cm high.
- Diet (e.g. ad libitum): A standard pelleted laboratory rodent diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24℃
- Humidity (%): 48 to 64%
- Air changes (per hr): Air exchange was maintained at approximately 19 air changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled to provide 12 hours artificial light (0700-1900 hours) in each 24-hour period.

IN-LIFE DATES: From 26 March to 23 April 1997

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% w/v methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared on a weekly basis. The required quantity of the test substance was mixed thoroughly with a small amount of 1% w/v aqueous methylcellulose to form a smooth paste. This was made up to volume with additional vehicle and further mixed using a high shear homogeniser. The resulting formulation was then divided into seven equal daily aliquots whilst being stirred magnetically.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 1.5, 5.0 or 15.0 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg/day
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 50 or 150 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
five male and five female
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance, a pharmaceutical intermediate, was administered by oral gavage once daily to groups of five male and five female rats for 28 consecutive days at dosage levels of 15, 50 or 150 mg/kg/day.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the treatment period all animals were observed prior to the doing and at regular intervals followings dosing each day. All signs of ill health, behavioural changes or toxicosis were recorded. All animals were additionally checked early in each working day and again in the late afternoon to look for dead or moribund animals. This allowed a post mortem examination to be undertaken during the working part of that day.

BODY WEIGHT: Yes
- Time schedule for examinations: The week prior to treatment (week -1), prior to dosing day (week 0) and subsequently at weekly intervals.

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

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 27
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, food was withdrawn overnight prior to collection of samples.
- How many animals: 40

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 27
- Animals fasted: Yes, food was withdrawn overnight prior to collection of samples.
- How many animals: 40

URINALYSIS: No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All superficial tissues were examined visually and by palpation, and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscrea was noted, with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastrointestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver was sectioned at intervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
HISTOPATHOLOGY: Yes
Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin wax (m.p. 56℃); sections were cut at 4 μm and stained with haematoxylin and eosin. A transverse section of each testis (left and right) and a full longitudinal section of each epididymis (left and right) was cut as near as possible to 2 μm and stained with PAS+-haematoxylin. Microscopic examination of the tests was made with reference to the stages of the cycle of the seminiferous epithelium.
Microscopic examination of prepared slides (from tissues indicated) was carried out for all rats from all groups.
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following sequence of statistical tests were used for bodyweight gains, organ weight and clinical pathology data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of values different from the mode were analysed by Fisher's exact test (Fisher, RA, 1950) followed by Mantel's test for a trend in proportions (Mantel, M. 1963). Otherwise:
Bartlett's test (Bartlett, MS, 1937) was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1% level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out followed by William's test (Williams, DA, 1971/1972) for a dose related response.
If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks (1952/1953) was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test, (Shirley, E, 1977)).
Covariate analysis of organ weight data (with final bodyweight as covariate) was also performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10% level of significance (Angervall, L and Carlstrom, E, 1963).
Significant differences between control animals and those treated with the test substance were expressed at the 5% (* p<0.05) or 1% (** p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
There were no unscheduled deaths during the study.
Transient post dose salivation was observed from Week 1 from animals receiving 50 and 150 mg/kg/day.
Males and females receiving 150 mg/kg/day and females receiving 15 or 50 mg/kg/day showed a superior bodyweight gain compared with controls.
Mean food intake for females receiving 150 mg/kg/day was greater than controls throughout the treatment period and for males only during the second half of the study.
Lower mean PCV, Hb and RBC values were noted for males and females receiving 150 mg/kg/day. Higher mean platelet values were noted for both sexes receiving 150 mg/kg/day. Lower mean total white cell count associated with a decrease in lymphocyte values was noted in males and females receiving 150 mg/kg/day. All of these differences were slight and of doubtful toxicological importance.
Higher mean total protein and globulin values and lower mean albumin and A/G ratio were noted for males and females receiving 150 mg/kg/day. Lower mean AP and GOT were noted for males and females receiving 150 mg/kg/day. Lower mean GPT values were noted for males and females receiving 150 mg/kg/day and males only receiving 50 mg/kg/day. A higher mean cholesterol value was noted for females receiving 150 mg/kg/day.
Mean liver weight was markedly higher for males and females receiving 150 mg/kg/day and females only receiving 50 mg/kg/day in comparison with controls.
Macroscopic post mortem examination revealed enlarged livers in all males and females receiving 150 mg/kg/day with the livers of 3/5 males showing accentuated lobular markings.
Treatment-related changes were reported in the liver. Centrilobular and midzonal hepatocyte hypertrophy was reported in males receiving 150 mg/kg/day and centrilobular hepatocyte hypertrophy was reported in males receiving 15 and 50 mg/kg/day. In females, centrilobular hepatocyte hypertrophy was reported in all treated groups. Dosage relationship was apparent in the severity of the change.

Effect levels

Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, 150 mg/kg/day was associated with a degree of effect of toxicological significance and the liver was suggested as a target organ.