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Toxicological information

Repeated dose toxicity: oral

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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other: GLP compliant, near-guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance code: L0212
Batch no. 01: L0212.01 (28% active ingredient)
Batch no. 02: L0212.02 (28% active ingredient)

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Age at study initiation: approximately 5 weeks
- Weight at study initiation: 156.3-178.8 g (males), 127.8-155.6 g (females)
- Fasting period before study: overnight fast before laboratory investigations and necropsy
- Housing: groups of 5 animals in stainless steel wire mesh cages suspended over cardboard-lined trays
- Diet: SQC Rat and Mouse Maintenance Diet No. 1, Expanded, Ground Fine (Special Diets Services Ltd., Witham), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days (palatability study), 11 days (toxicity study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): minimum 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION:
The test article was mixed with glycerol to aid the mixing and distribution of the test article in the diet (volume of glycerol about 1% of final diet mix). Formulations were then mixed with a small amount of powdered diet to produce a pre-mix which was then added to the remaining diet and mixed for a minimum of ten minutes. The diet for the group 2 animals was achieved by dilution of the group 3 formulation. A similar amount of glycerol was added to the control diet. The test diets were prepared weekly, reserve samples of 100 to 200 g being retained from each batch.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from diets prepared in weeks 1 and 13 for group 2, 3 and 4 were taken for analysis of achieved concentration. These samples were stored deep frozen before despatch to the sponsor for analysis. Reserve samples taken in weeks 4 and 8 were also despatched to the sponsor for analysis.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
25 (group 2), 250 (group 3), 2500 (group 4) mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on range finding study
- Rationale for animal assignment: the animals were randomly allocated to the palability study. The toxicity phase animals were assigned to treatment groups using a randomisation procedure based on stratified body weights during the acclimatisation period. Group mean body weights were calculated to ensure no unacceptable differences between groups. Treatment group positions in the battery were assigned using a set of random letter permutations.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily during palatability phase; before treatment on first day of the study, at weekly intervals thereafter and at necropsy (toxicity phase)

FOOD CONSUMPTION AND COMPOUND INTAKE:
- The amount of food consumed by each cage of animals was determined daily during the palatability phase and weekly during the toxicity phase

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-dose (all animals), week 13 (control and high dose animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13 (all animals), week 14 (control and high dose animals, APTT and PT only)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- Parameters examined were: haemoglobin concentration, mean cell volume, red blood cell count and indices (mean cell haemoglobin, mean cell haemoglobin concentration, packed cell volume), total and differential white blood cell count, platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: all
- Parameters examined were: aspartate aminotransferase/glutamate oxaloacetate transaminase, alanine aminotransferase/glutamate pyruvate transaminase, alkaline phosphatase, sodium, chloride, inorganic phosphorous, urea, potassium, calcium, glucose, total bilirubin, creatinine, albumin, total cholesterol, total protein, albumin/globulin ratio
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Full internal and external examination, organ weights (adrenals, liver, kidneys, testes (and epididymides))

HISTOPATHOLOGY: Yes
Tissues examined were: adrenals, brain (including brain stem), colon, heart, ileum, aorta, caecum, duodenum, jejunum, kidneys, liver, lymph nodes (mandibular and mesenteric), pancreas, stomach, thymus, trachea, uterus, lungs (with mainstem bronchi), oesophagus, ovaries, pituitary, rectum, sciatic nerve, spleen, sternum, testes with epididymides, thyroids (with parathyroids), urinary bladder, all gross lesions
Statistics:
Body weights, body weight gain and organ weights were analysed using analysis of variance (ANOVA). Comparisons between each treated and control group were made using Dunnett's test. Regression analysis for dose-response was also performed.
Haematology and clinical chemistry parameters were analysed using ANOVA, Dunnetts's test and regression analysis. Total bilirubin (males) and prothrombin time (males) were analysed non-parametrically using the Kruskel-Wallis non-parametric ANOVA with the protected Wilcoxon rank-sum test. The Terpstra-Jonckheere test for dose-response was performed. Data for GOT(AST) and GPT(ALT) were log-transformed prior to analysis.
All tests were carried out at 0.1, 1 and 5% levels of significance. Males and females were analysed separately.

Results and discussion

Results of examinations

Details on results:
PALATABILITY PHASE
All animals gained weight and food consumption was as expected for animals of this age. The test diet was therefore considered to be palatable at the highest concentration likely to be used during the study.

CLINICAL SIGNS AND MORTALITY
There were no deaths; all animals survived to study termination in apparent good health.
From week 1 all group 4 animals were noted to be blue/green in color. This is considered to be due to absorption of the test article. Dark blue/green faeces were noted from week 3 in all treated groups.

BODY WEIGHT AND WEIGHT GAIN
Group 4 male and female animals gained less weight than the control animals over the 13 week treatment period (p<.01 for males, p<.05 for females).By week 13 group mean body weights of group 4 males and females were about 12% less than those of the control groups.
There was no effect on the body weight of group 2 and 3 animals.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was variable in all groups throughout the study, however, group 4 male animals generally consumed more (by about 7%) than the control animals. There was no apparent effect on the food consumption of group 4 females or group 2 and 3 animals, males or females.
Calculated compound consumption was generally in good agreement with the nominal dose levels.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopic findings in group 4 animals at week 13 to suggest any effect of treatment. The eyes of group 2 and 3 animals were therefore not examined.

HAEMATOLOGY
At week 13, the mean cell haemoglobin concentration (MCHC) was higher (p<.001) and packed cell volume (PCV) lower (p<.05) in group 4 males compared to controls. The red blood cell count and haemoglobin concentration were lower in a few animals. In group 3 males MCHC was higher (p<.05) than controls.
At week 13 prothrombin time (PT) and activated partial thromboplastin time (APTT) were increased slightly in several group 4 male animals. As a result group mean PT and APTT were greater than those of the control by 13% and 17% respectively. In the group 4 females group mean PT was 7% greater than that of the control group (p<.05); APTT was comparable to that of the control group. In group 3 males APTT was greater than that of the control group by 8%. With the exception of the PT increase in group 4 females these differences did not achieve statistical significance.
There was no apparent effect in group 3 females or group 2 animals.
The plasma of several of the high dose animals was visibly green in colour and it was considered that the green colour of the plasma might be interfering with the analysis. Blood samples were therefore taken fom control and group 4 animals before necropsy and the analysis of PT and APTT repeated using a manual method. Group 4 male PT was still greater than that of the control (by 13%). APTT values for group 4 males were similar to the controls. There were no other changes in haematology parameters.

CLINICAL CHEMISTRY
At week 13 the group mean urea level of group 4 males was greater than that of the control group by 13% but this was not statistically significant.
There were apparent changes in calcium, total bilirubin, protein and cholesterol in group 4 animals. However, the plasma of several group 4 animals was visibly green and it was considered possible that the green colour of the plasma may be affecting the analysis. The effect on the analysis of calcium, phosphorous, glucose, urea, total bilirubin, creatinine, total protein, albumin and total cholesterol was investigated. The presence of test article in the pleura was shown to interfere with the analysis of calcium and total bilirubin, protein and cholesterol. Meaningful evaluation of these parameters is therefore not possible.

ORGAN WEIGHTS
There were no changes considered to be related to treatment. Changes in relative kidney and testes weights of group 4 males end relative liver weights of group 4 males and females are considered to reflect the effect on body weight in this group.

GROSS PATHOLOGY
Most control animals were unremarkable at necropsy. In groups 2, 3 and 4 there was a green discolouration involving progressively more organs. In group 2, the green discolouration involved mainly the contents of the large intestine. In group 3 most of the gastrointestinal tract was green, the liver and ovaries were also green. In group 4 the whole animal was green.

HISTOPATHOLOGY
Histopathologically, changes were seen in the gastrointestinal tract of group 3 and 4 animals and in the kidneys of group 4 females.
Squamous hyperplasia of the limiting ridge of the forestomach was seen in group 4 animals, this effect is however not relevant for humans. A low grade caecitis in the large intestine in some cases accompanied by colitis or proctitis, was observed in some group 3 and 4 animals. There were no changes in the gastrointestinal tract of group 2 animals.
Minor hyaline droplet accumulation in the proximal tubular epithelium was observed in some group 4 female animals and was considered to be related to treatment. There was no increase in hyaline droplets in the kidneys of group 2 and 3 females. The high level of hyaline droplets in control males masked the possible detection of any effect in treated males.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
25 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: caecitis accompanied by colitis or proctitis in the large intestine of animals treated at 250 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
250 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: increase in hyaline droplet accumulation in the proximal tubular epithelium of the kidney in females in the high dose group

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion