Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 Jan to 03 Jun 1999
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:
1999
Report Date:
1999

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 (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch No.: 8-UAD-015
Purity: Not specified

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: six to seven weeks old
- Weight at study initiation: the males weighed 177 to 220 g, the females weighed 159 to 196 g
- Fasting period before study:
- Housing: :The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): The animals were allowed free to food. A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK) was used.
- Water (e.g. ad libitum): The animals were allowed free to water. Mains water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: eleven days

ENVIRONMENTAL CONDITIONS
- Temperature (℃): 21±2
- Humidity (%): 50±15
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The formulations are stable for at least fourteen days.
- Concentration in vehicle: 37.5, 125, 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.
Duration of treatment / exposure:
twenty-eight consecutive days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
150, 500 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends and public holidays.

FUNCTIONAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and on Days 4, 12, 21 and 27 all animals were observed for signs of functional/behavioral toxicity. On Day 27 functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was recorded for each cage group at weekly intervals throughout the study.

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: Yes
- Time schedule: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (Day 28)
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all animals
- Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), Total leucocyte count (WBC), Differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophiIs (Bas)), Platelet count (PLT), Reticulocyte count (Retic) (Cresyl blue stained slides were prepared but reticulocytes were not assessed), Prothrombin time (CT), Activated partial thromboplastin time (APTT) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (Day 28)
- Animals fasted: No
- How many animals: all animals
-Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (CI-), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total Cholesterol (Chol) and Total bilirubin (Bili) were examined.

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
- Parameters checked in table [No.?] were examined.

Sacrifice and pathology:
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone (Sagatal, 60 mg/mL) followed by exsanguination.
- Organ Weights: The following organs removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: Adrenals, Kidneys, Testes, Brain, Liver, Thymus, Epididymides, Ovaries, Heart, Spleen.
- Histopathology: Bone&bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Gross lesions, Heart, Ileum, Jejunum, Ovaries, Prostate, Rectum, Sciatic nerve, Seminal vesicles, Spinal cord (cervical), Spleen, Stomach, Testes, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Thymus, Thyroid/parathyroid, Trachea, Urinary bladder and Uterus from all control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The liver and spleen from all 150 and 500 mg/kg/day dose group animals were also processed.
Statistics:
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ”U” test.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.
Probability values (p) are presented as follows: p<0.001***, p<0.01**, p<0.05*, p ≥ 0.05 (not significant).

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:
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
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
Mortality
There were no deaths during the study.

Clinical Observations
No clinically observable signs of toxicity were detected in test or control animals throughout the study period.
Females treated with 1000 mg/kg/day showed incidents of red/brown staining of the fur from Day 10 onwards. Fur staining became more pronounced in these animals as the study progressed and by Day 27 red/brown staining on the head was recorded, evident in all females by Day 27. In the absence of any evidence to suggest an adverse health effect in these animals, however, the high incidence of staining was considered to be entirely fortuitous and unrelated to treatment with the test material.
There was no evidence of fur staining in 1000 mg/kg/day males or in animals of either sex treated with 500 or 150 mg/kg/day.

Functional Observations
- Behavioural Assessments: Detailed behavioural assessments revealed no treatment-related intergroup differences. All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were, therefore, of no toxicological importance.
- Functional Performance Tests: There were no treatment-related changes in the functional performance parameters measured. Statistical analysis of the data revealed no significant intergroup differences.
-Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were, therefore, of no toxicological importance. Statistical analysis of the quantitative data revealed no significant intergroup differences.

Bodyweight
All animals showed normal gains in bodyweight throughout the study period. Bodyweight gain in test animals was similar to that of controls. Statistical analysis of the data revealed no significant intergroup differences.

Food Consumption
No adverse effect on food consumption was detected during the study. Food efficiency (the ratio of bodyweight gain to dietary intake) was similar to that of controls.

Water Consumption
Daily visual inspection of water bottles revealed no intergroup differences.

Haematology
There were no treatment-related changes in the haematological parameters measured.
A statistically significant reduction in haemoglobin was detected for all male treatment groups compared with that of controls. The level of statistical significance was minimal (p<0.05) and, in the absence of a convincing dose-related response or other haematological changes, the intergroup differences were considered to be attributable to slightly elevated control values rather than an effect of treatment. A slight but statistically significant (p<0.05) increase in platelet count was detected for 1000 mg/kg/day females but in isolation this was considered to be of no toxicological importance.

Blood Chemistry
There were no treatment-related changes in the blood chemical parameters measured.
Statistically significant increases in plasma albumin were detected for all female treatment groups and males treated with 1000 or 500 mg/kg/day; total plasma protein was also elevated for these male treatment groups when compared with controls. The dose response relationship was unconvincing and, in the absence of any other evidence to suggest dehydration, the intergroup differences were considered to be without toxicological importance. A similar unconvincing dose response was evident for plasma chloride concentration with a slight increase seen in this parameter for all female treatment groups. The level of statistical significance was minimal (p<0.05) and, in isolation, was considered to be fortuitous. The remaining statistically significant intergroup difference detected was confined to an increase in plasma alkaline phosphatase for 500mg/kg/day males, but without a convincing dose relationship, this was considered not to be toxicologically important.

Pathology
- Organ Weights: All female treatment groups showed a statistically significant increase in liver weight, relative to bodyweight when compared with controls. Absolute liver weight was also elevated in treated females but statistical significance was not achieved for 150 mg/kg/day animals. Many of the individual values, both absolute and relative to bodyweight were outside of the respective normal ranges for rats of the strain and age used. In the absence of any histopathological evidence to suggest an adverse liver effect, however, the increases were probably a result of adaptive change and, as such, did not represent an adverse effect on the health of the animals. There were no significant intergroup differences in liver weight for the male treatment groups.
- Necropsy: No treatment-related macroscopic abnormalities were detected at terminal kill. The incidental finding recorded at terminal kill was confined to a control male and was identified as dark areas on the left lobe of the lungs. This was consistent with a normal low incidence finding in laboratory maintained rats and was incidental.
- Histopathology: No treatment-related changes were observed. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

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: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in a treatment-related liver weight increase for females treated with 1000, 500 and 150 mg/kg/day. No such changes were observed amongst the male treatment groups and the NOEL was, therefore, considered to be 1000 mg/kg/day for males.
The liver weight increase in the female treatment groups was not associated with histopathological changes and was considered not to represent an adverse health effect. The "No Observed Adverse Effect Level" (NOAEL) is, therefore, considered to be 1000 mg/kg/day for either sex.