416-730-1

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline conform

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were received from Charles River (UK) Ltd., Margate, Kent, England. The rats were 28 ± 1 days old, in a weight range of 70 - 82 g on arrival. A thirteen day acclimatisation period was allowed between delivery of the animals and start of treatment.
All rats were initially caged, as far as possible, in groups of five according to sex in metal cages with wire mesh floors.
A standard pelleted laboratory rodent diet (Special Diet Services Rat and Mouse Maintenance Diet) and drinking water was provided ad libitum, except as noted under "CLINICAL PATHOLOGY". The batches of diet used for the smdy had been analysed for nutrients, possible contaminants and micro-organisms.
Results of the routine physical and chemical examination of drinking water at source, as conducted, usually weekly by the supplier, were made available to Himtingdon Research Centre Ltd. as quarterly summaries.
Animal room temperature was controlled within the range 17.5 to 25°C and relative humidity was controlled in the range 39 to 71 % RH. These parameters were continuously monitored using a Kent Clearspan M105 7-day chart recorder. Air exchange was maintained at approximately 19 air changes per hour and lighting was controlled to provide 12 hours artificial light (0700-1900 hours) in each 24-hour period.
The health status of all animals was monitored, by daily observation throughout the acclimatisation period, to ensure that the rats selected for final assignment to the study were satisfactory.
Four days prior to the start of treatment each animal was weighed and sixty rats were randomly allocated to four groups, two groups consisting of ten males and ten females and two groups consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.
Each rat was identified within each cage by ear-punch and individually by tail mark (tattoo).
Following the commencement of treatment spare animals were removed from the study. No further investigations were performed on these animals.
The cages (each containing five rats) were distributed in batteries in such a manner that possible environmental influences arising from their spatial distribution were equilibrated, as far as possible, for all treatments.
Each cage was identified by a coloured label according to group. Each label displayed the study schedule number, cage number, sex, individual animal numbers and the initials of the Study Director and Home Office licensees.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
The test substance was weighed out and mixed with the vehicle (1% w/v aqueous methylcellulose) using a high shear homogeniser. For each concentration this process was repeated. Formulations were prepared freshly each day.

The chemical stability and, homogeneity of test substance formulations were assessed prior to the start of treatment and, concentration analyses of formulations prepared for administration on Days 1 and 22 were performed by the Department of Analytical Chemistry at HRC

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chemical stability and, homogeneity of test substance formulations were assessed prior to the start of treatment and, concentration analyses of formulations prepared for administration on Days 1 and 22 were performed by the Department of Analytical Chemistry at HRC.

Duration of treatment / exposure:

28d

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
15, 150 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10 for high dose and control
5 for low and mid dose

Control animals:

yes, concurrent vehicle

Details on study design:

The high dosage was selected on the basis of available toxicity data and a preliminary oral toxicity investigation. A dosage of 1000 mg/kg/day is the limit level for this study design.

Following the 4 week treatment period, five male and five female animals from Groups 1 and 4 (lowest animal numbers were selected) and survivinganimals of Groups 2 and 3 were killed on Day 32 (post-treatment sacrifice). These rats were dosed until the day prior to sacrifice. The remaining animals of Groups 1 and 4 were retained for a 2 week post-treatment observation period and were killed on Day 46 (recovery sacrifice); these rats received their last dose on Day 28.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 3 x daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- The quantity of food consumed in each cage was measured at weekly intervals throughout the study

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. Different sequence of statistical tests was used for bodyweight gains, food consumption (using weekly cage totals), organ weight and clinical pathology data.

Results and discussion

Results of examinations

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

Water consumption and compound intake (if drinking water study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

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

Details on results:

The only clinical observation during the treatment period of the study was yellow-coloured faeces on the cage tray paper under the cages of rats receiving 1000 mg/kg/day. This incidental finding was considered to be due to the presence of the test substance in the faeces and not a manifestation of toxicity.

After the recovery period, kidney weight (bodyweight adjusted) was statistically significantly lower than the control for male rats treated at 1000 mg/kg/day and higher than control adrenal weights were recorded for female rats treated at 1000 mg/kg/day. In the absence of any treatment-related effects on these organs at Week 5 these small differences from control were considered to reflect variation.

Effect levels

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It was concluded that 1000 mg/kg/day represents the no-observed effect level (NOEL) for the test substance in the rat. Labelling with the R48 risk phrase according to the EEC Council Directive 79/831/EEC, Annex VI Part 11(D) as described in Commission Directive 93/21/EEC is not required.

Executive summary:

The substance was administered by oral gavage, once daily, to three groups of rats for a minimiun for twenty eight consecutive days, at dosage levels of 15, 150 or 1000 mg/kg/day. The test material was prepared as suspensions in 1% w/v aqueous methylcellulose (1% MC) at concentrations of 0.15, 1.5 or 10% w/v and was administered at a dosage volume of 10 ml/kg/day. Control animals received the vehicle (1% MC) alone at the same dose volume (10 ml/kg/day).

All rats of Groups 2 and 3 (15 and 150 mg/kg/day respectively) and five males and five females from each of Groups 1 and 4 (Control and 1000 mg/kg/day respectively) were killed following the fourweek treatment period (Day 32). The remaining animals (five males and five females from Groups 1 and 4) were retained for a two-week recovery period, following which, they

were also killed (Day 46).

Bodyweights, food consumption and clinical observations were recorded during the study. Blood and urine samples were taken from all rats shortly prior to termination following the four-week treatment and two-week recovery periods. All animals were killed and subsequently examined macroscopically; specified tissues were then prepared for histopathological examination.

There were no changes seen in the parameters measured in this study namely clinical signs, bodyweights, food consumption, clinical pathology, organ weight analysis, macroscopic or microscopic pathology.