Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

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:
From 26 APR 1994 to 06 JUN 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 474)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
only 1000 instead of 2000 immature erythrocytes were evaluated per animal
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
only 1000 instead of 2000 immature erythrocytes were evaluated per animal
GLP compliance:
yes (incl. certificate)
Remarks:
in accordance with Directive 88/320/EEC
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan U.K. Ltd., blackthorn, Bicester, Oxon, U.K.
- Age at study initiation: 5-8 weeks
- Weight at study initiation: males: 21-30 g; females: 20-25 g
- Assigned to test groups randomly: yes
- Housing: in groups up to five by sex
- Diet: Rat and Mouse Expanded Diet No. 1 (Special Diet Services Ltd. Witham, Essex, U.K.), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 44-50
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: arachis oil
- Concentration of test material in vehicle: 125, 250 or 500 mg/ml
- dose volume: 10 ml/kg

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- test material was freshly prepared as a suspension at the appropriate concentration in arachis oil
- analysis of concentration, homogeneity and stability was not performed

APPLICATION:
- single intraperitoneal application
Duration of treatment / exposure:
1250 and 2500 mg/kg group: 24 hrs
5000 mg/kg group: 24, 48 and 72 hrs
Frequency of treatment:
single
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
1250, 2500, 5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females per dose and exposure duration
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 50 mg/kg
- Exposure duration: 24 hrs

Examinations

Tissues and cell types examined:
bone marrow cells from the femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- maximum recommended dose level and two lower dose levels

DETAILS OF SLIDE PREPARATION:
- femur was dissected from each animal, aspirated with foetal calf serum and bone marrow smears were prepared following centrifugation and re-suspension. Smears were air-dried, fixed in absolute methanol and stained with May-Grünwald/Giemsa

METHOD OF ANALYSIS:
- blind examination with light microscopy
- scoring of micronucleated cells per 1000 polychromatic erythrocytes per animal
- normochromatic erythrocytes associated with 1000 erythrocytes were counted ans scored for incidence of micronuclei
- calculation of polychromatic to normochromatic erythrocytes

Evaluation criteria:
- a statistically significant increase in the number of micronucleated polychromatic erythrocytes in the treatment groups in comparison to the control group is evaluated as positive mutagenic response
- a positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio is shown to be statistically significant from the concurrent vehicle control group
Statistics:
- Student's t-test
- one way analysis of variance

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-5000 mg/kg bw, i.p; 500-2000 mg/kg bw, oral
- Clinical signs of toxicity in test animals: hunched posture and fur stained orange; one premature death at 2000 mg/kg bw i.p., which was considered to be due to poor dosing technique rather than to toxicity of the test material



RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): group means for control groups: 1.46 - 1.83; treatment groups did not deviate significantly

Any other information on results incl. tables

- no premature deaths in any dose group

- fur stained with test material in all animals treated with the test item

- hunched posture in some animals at 5000 mg/kg bw

- one animal with lethargy at 5000 mg/kg bw

There was no statistically significant increase in the frequency of micronucleated PCE's and in the PCE/NCE ratio in any dose group when compared to the concurrent vehicle control groups.

The positive control goup showed a marked increase in the incidence of micronucleated PCE.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item was considered to be non-genotoxic under the conditions of this in vivo micronuclei assay.
Executive summary:

Genetic toxicity of the test item has been investigated in an in vivo micronucleus assay in male and female ICR mice (5 per sex and group). The test item was administered at the maximum recommended dose level of 5000 mg/kg bw, with 2500 and 1250 mg/kg bw as the two lower dose levels. Animals were killed 24, 48 or 72 hours later. Evaluation of polychromatic (PCE) or normochromatic (NCE) erythrocytes did not reveal any evidence of an increase in the incidence of micronucleated cells. No significant change in the PCE/NCE ratio was observed after dosing with the test item, however, clinical signs were observed in animals dosed with the test item and as such are evidence of systemic exposure. The positive control material produced a marked increase in the frequency of micronucleated PCE. The test material was considered to be non-genotoxic under the conditions of the test.