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:
2010/2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1997
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SR531
- Physical state: pale yellow liquid
- Analytical purity: not given
- Lot/batch No.: KTE0864
- Expiration date of the lot/batch: 2010-12-16
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 6-10 weeks
- Weight at study initiation: 24-30g
- Assigned to test groups randomly: yes
- Housing: groups of up to 7 mice in solid floor polypropylene cages
- Diet (e.g. ad libitum): Harlan Teklad 2014 Global Certified Rodent Diet ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: solubility of the test substance and availability of historical control data
- Concentration of test material in vehicle: 7.5 - 30mg/ml

Frequency of treatment:
once
Post exposure period:
24h (75, 150, 300mg/kg), 48h (300mg/kg)
Doses / concentrations
Remarks:
Doses / Concentrations:
75, 150, 300 mg/kg
Basis:

No. of animals per sex per dose:
7 per dose and evaluated time point, 5 in the positive control group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral (gavage)
- vehicle: destilled water
- Doses / concentrations: 50mg/kg b.w. at a concentration of 5mg/ml

Examinations

Tissues and cell types examined:
bone marrow (femur)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the micronucleus test. With no evidence of toxicity initially via the oral route at 2000mg/kg b.w., it was considered necessary to investigate the intraperitoneal route of administration. The following doses were tested: 1000, 600, 400, 300mg/kg b.w. Animals were observed one hour after dosing and subsequently once daily for two days. Severe clinical signs were observed at and above 400 mg/kg as follows: hunched posture, ptosis, lethargy, ataxia, pilo-erection, decreased respiratory rate, laboured respiration, hypothermia, emaciation, dehydration, and splayed gait. The clinical signs observed in animals dosed via the intraperitoneal route at 300 mg/kg were as follows: hunched posture and ptosis. Therefore, with evidence of excessive toxicity at and above 400 mg/kg, the main test was performed using the maximum tolerated dose level of 300 mg/kg, with 150 and 75 mg/kg as the two lower dose levels. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice.

DETAILS OF SLIDE PREPARATION:
Immediately following termination, both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/ Giemsa, allowed to air-dry before a cover slip was applied using mounting medium.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blindly using light microscopy at x1 000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes ( PCE-blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
Evaluation criteria:
A positive mutagenic response would be demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to the vehicle control group.

A positive response for bone marrow toxicity would be demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part Ill (1989). The data was analysed following a (x+1)^(1/2) transformation 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:
- hunched posture, ptosis, pilo-erection, and tip-toe gait at and above 75mg/kg; - modest statistically significant decrease in the PCE/NCE ratio at 300mg/kg after 24h (not statistically significant decrease at 300mg/kg after 48h and 150mg/kg)
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative