Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Jan 2002-05-Feb 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in 2002 according to OECD Method 474 and EU Annex V test B12and in accordance with GLP. Study material is well characterized.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test material is a extremely light colored powder which was received at testing laboratory on 24 October 2001 and stored at room temperature in the dark.

Test animals

Species:
mouse
Strain:
other: albino Cr1:CD-lTM(ICR)BR strain mice
Sex:
male
Details on test animals and environmental conditions:
At the start of the study the mice were in the weight range of 24 to 30 grams and were five to eight weeks old. Free access to mains drinking water and food was allowed throughout the study. The temperature and relative humidity were set to achieve limits of 19 to 25 C and 30 to 70% respectively. The rate of air exchange was at least 15 changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours of darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Arachis oil
Details on exposure:
Doses of test material: 375, 750 or 1500 mg/kg were selected based on a range finding toxicity study.
Duration of treatment / exposure:
The mice were dosed only once and one group of mice from each dose level (375, 750 or 1500 mg/kg) were killed by cervical dislocation 24 hours following treatment and a second group dosed with 1500 mg/kg was killed after 48 hours.
Frequency of treatment:
Groups, each of seven mice, were dosed once only via the oral route with test material.
Post exposure period:
One group of mice from each dose level (375, 750 or 1500 mg/kg) were killed by cervical dislocation 24 hours following treatment and a second group dosed with 1500 mg/kg were killed after 48 hours. All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
375 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
750 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Male: 1500 mg/kg; No. of animals: 7; Sacrifice time: 48 hours
Male: 1500 mg/kg; No. of animals: 7; Sacrifice time: 24 hours
Male: 750 mg/kg; No. of animals: 7; Sacrifice time: 24 hours
Male: 375 mg/kg; No. of animals: 7; Sacrifice time: 24 hours
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
A positve control group of five animals were dosed at 50 mg/kg using cyclophosphamide.
Two groups of severn animals were used as a vehicle control group (Arachis oil) with one group terminated 24 hours and the second 48 hours after dosing.

Examinations

Tissues and cell types examined:
Bone marrow from the femurs of each animal used for smears. Cell types examined for the presence of micronuclei in Polychromatic (PCE) and normochromatic (NCE) erythrocytes.
Details of tissue and slide preparation:
Immediately following termination, bone marrow smears were prepared from sections of the femurs from each animal and stained with May-Griinwald/Giemsa.Stained bone marrow smears were coded and examined blind using light microscopy at xlOOO magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE- blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group. A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of rnicronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group. A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group
Statistics:
Group means and standard deviations for the PCE/NCE ratios were calculated for the data. The data was analysed using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2000 mg/kg
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Range finding study: The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main study. Adequate evidence of test material toxicity was demonstrated via the oral route of administration, therefore, this was selected for use in the main study. The maximum tolerated dose (MTD) of the test material, 1500 mgkg, was selected for use in the main study, with 375 and 750 mgkg as the lower dose levels.

Toxic signs: In the range finding study, one male animal dosed orally at 2000mg/kg bw died following treatment. Clinical signs observed at and above 1000mg/kg bw in the range-finding and main studies included hunched posture, lethargy, tremors, laboured respiration and piloerection.

No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. However, small dose-related decreases in PCE/NCE ratios were observed, these and the presence of clinical signs were taken to indicate that systemic absorption had occurred. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

Observations:
There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at 1500 mg/kg in both the 24 and 48-hour groups, these were as follows: hunched posture, lethargy and body tremors.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test. The test material was considered to be non-genotoxic under the conditions of the test.
Executive summary:

The study is considered valid and assigned a reliability rating of 1. Micronucleus in vivo:

No statistically significant decreases in the PCE/NCE ratio were observed in the 24- or 48 -hour test material dose groups when compared to their concurrent control groups. However, small dose-related decreases in PCE/NCE ratios were observed, these and the presence of clinical signs were taken to indicate that systemic absorption had occurred. The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test. The test material was considered to be non-genotoxic under the conditions of the test.