Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Dec 1990 - 16 Jan 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): T-4113
- Physical state: white powder
- Analytical purity: >96%
- Lot/batch No.: 5004
- Expiration date of the lot/batch: October 1991
- Storage condition of test material: under ambient conditions in the dark

Test animals

Species:
rat
Strain:
other: Crl: CD SD BR VAF/Plus
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd., Margate, Kent, England
- Age at study initiation: 28 ± 1 days
- Weight at study initiation: range of 66 - 80 g
- Housing: in groups of 5
- Diet: Biosure LAD 1 Diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 50 -60
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Dec 1990 To: 16 Jan 1991

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was ground in a mortar with a small amount of the vehicle (1% methylcellulose) until a smooth paste was formed. The formulation was then gradually made up to volume and mixed using a high shear homogeniser. A series of formulations were prepared freshly each day. Prior to dosing the test substance formulations were mixed by inversion (x20) and subsequently using a magnetic stirrer for a period of at least 10 minutes before dosing commenced. Dosing was completed within one hour of the commencement of stirring.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of test substance in a representative sub-sample was quantified by high perfomrance liquid chromatography using ultra-violet detection, to determine concentration and chemical and physical stability. For calibration a standard solution was prepared by dissolving an accurately weighed quantity of test substance and preparing serial dilutions. The results were within 5% of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.1, 1, 10% (w/v)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10, 100, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected based on a 7-day preliminary oral toxicity study

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing and subsequently at weekly intervals

FOOD CONSUMPTION:
- Food consumption for each cage: measured at weekly intervals

WATER CONSUMPTION: Yes
- Time schedule for examinations: first 14 days by visual appraisal, since treatment-related effect was suspected, water consumption was measured by gravimetric analysis from week3 on

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.2] were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table3)
HISTOPATHOLOGY: Yes (see table4)
Statistics:
All statistical analysis were carried out seperatedly for males and females. Bodyweight data were analysed using weight gains.
The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data:
1) If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of values different from the mode was analysed by appropriate methods. Otherwise:
2) Bartlett's test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level)heterogeneity was found, a logarithmic transformation was tried to see if a more variance structure could be obtained.
3) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
4) Analysis of variance were followed by student's t-test and Williams test for a dose-related response, although only the one thought more appropriate for the response pattern observed reported. The Kruskal-Wallis analysis were followed by the non-parametric equivalents of the t test and Williams test (Shirleys test).
Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, analysis initially involved a correlation analysis between organ weights and final body weight. For organs where a correlation at the 10 % level of significance was established, analysis of organ weight data was performed using adjusted organ weights by analysis of covariance with final bodyweight as covariate. Where a correlation between organ weight and bodyweight was not established the organ weight analysis was carried out using routine analysis of variance on unadjusted organ weights.

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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day females: 24% increase, 1000 mg/kg bw/day females: 35% increase, 100 + 1000 mg/kg bw/day males: slight decrease (20%), non adverse
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
decreased lymphocyte and total white blood cell count in one female at 1000 mg/kg bw/day, non adverse
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day - female: adjusted adrenal and liver weights significantly increased, males: adjusted liver weights slightly increased, non adverse
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Clinical findings were only observed after withdrawal of blood (slight pallor of extremities, slight to moderate swelling of the right eye in three animals).

BODY WEIGHT AND WEIGHT GAIN
No changes in body weight gain were observed.

FOOD CONSUMPTION
No apparent differences were observed.

WATER CONSUMPTION
A slight disturbance of water consumption at 100 and 1000 mg/kg/day, but this was considered toxicologically not relevant.

HAEMATOLOGY
A decreased lymphocyte and total white blood cell count was observed at 1000 mg/kg/day in 1 female and where therefore considered to be of minor importance.

CLINICAL CHEMISTRY
No treatment related effects were found.

ORGAN WEIGHTS
An increase in relative liver weights and adrenal weights were found for females. Increase of liver weights were associated with hepatic enzyme induction as an adaptive response. No histological findings were observed in the adrenal glands, therefore the weight change was considered toxicologically not relevant.

HISTOPATHOLOGY: NON-NEOPLASTIC
Only one female showed minimal centrilobular hepatocyte enlargement, which is usually associated with increased hepatic activity and can be considered to be an adaptive response.

Effect levels

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: no adverse effects observed up to and including highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion