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:
supporting study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well reported GLP study, comparable to guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
other: Crl: NMRI, BR mice (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, Sulfeld, Germany
- Age at study initiation: 8 - 9 weeks old
- Acclimation period: 13 days

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
9 mg NaCl, 0.85 Polyoxyl-50-stearate (Myrj 52) at 1 ml Aqua p.i.
amount of drug substance per unit: 50 mg/mL
Details on exposure:
dose-range finding experiment:
- One male and one female mice per dose were treated once intraperitoneally with doses of up to 2000 mg/kg bw. All treated animals were without clinical findings during an observation period of 48 hours after application. However, 72 hours after application both animals treated with 1500 mg/kg bw showed slight apathy. At the dose of 2000 mg/kg bw the female mouse showed slight apathy and the male mouse died. Therefore, for the bone marrow micronucleus test 2000 mg/kg bw was chosen as the high dose group. Additionally, 1000 and 500 mg/kg bw were chosen as mid and low doses.
Duration of treatment / exposure:
single administration
Frequency of treatment:
single administration
Post exposure period:
24 h (negative control and all treatment groups)
48 h (positive control and high-dose group)
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, and 2000 mg/kg bw
Basis:
nominal conc.
application volume: 10 to 40 ml/kg
No. of animals per sex per dose:
5 per sex per dose for the 24 hour sampling time (vehicle control, 500 mg, 1000 mg, 2000 mg and positive control cyclophosphamide) plus
5 per sex per dose for the 48 hour sampling time (vehicle control, and 2000 mg/kg bw) were evaluated;
some more animals (all together 13 mice per sex) were treated in the high dose group to obtain 10 animals per sex for assessment.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (30 mg/kg bw)

Examinations

Tissues and cell types examined:
Erythrocytes; femur bone marrow smears were prepared and stained using May-Gruenwald and Giemsa solutions.
Evaluation criteria:
- frequency of micronucleated poly- and normochromatic bone marrow erythrocytes (= genetic endpoint chromosome/genome mutations)
- ration of poly- to normochromatic erythrocytes (= indicates bone marrow cytotoxicity)
- additionally, clinical signs were examined

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2000 mg/kg bw: apathy, increasing with time; one male died; a statistically significant decrease in the ration of PCE/NCE was seen in all dose groups for the 24 h time point as well as in the high dose group for the 48h time point
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The male and female animals in all dose groups, treated with 500, 1000, or 2000 mg/kg bw, showed neither a biologically relevant nor statistically signficant increase in micronucleated PCE and NCE as compared to the negative control at either of the two sampling times (24 and 48 hours). The decrease of the PCE/NCE ratio indicated bone marrow depression induced by the test substance. The positive control data were well in the expected historical range. However, in the vehicle control group the micronucleus frequency slightly exceeded the historical control data. But since the values are well within the published spontaneous rates (0.4 -3.8 per 1000 PCEs) this effect is considered as biological fluctuation.

Applicant's summary and conclusion

Executive summary:

To investigate the potential of the test item to induce chromosomal breakage (structural chromosomal aberrations) or misdistribution of chromosomes (aneuploidy), a bone marrow micronucleus test was performed on mice. Ten male and ten female mice in the high dose group and 5 males/females in the mid and low dose group were treated intraperitoneally with single doses of 500, 1000, or 2000 mg/kg bw. Control animals (10 males/ 10 females) were given the vehicle (Myrj 52) in the same manner. The test item showed neither a biologically relevant nor statistically signficant increase in micronucleated polychromatic (PCE) and normochromatic erythrocytes (NCE) as compared to the negative control at either of the two sampling times (24 and 48 hours). The decrease of the PCE/NCE ratio indicated bone marrow depression induced by toxicity of the test substance. One male mouse died in the high-dose group. Thus, the test substance was negative in the mouse bone marrow micronucleus assay up to the recommended and toxic dose level of 2000 mg/kg bw.