Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Kn 172

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
physiol. saline
Duration of treatment / exposure:
24, 48 hours
Frequency of treatment:
single
Doses / concentrations
Remarks:
Doses / Concentrations:
7.5, 15, 30 mg/kg bw
Basis:
other: vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
The positive control used in the micronucleus test was cyclophosphamide (CAS no.
50-1 8-0; Endoxan) dissolved in physiological saline
dosed as a single intraperitoneal injection of 50 mglkg body weight.

Examinations

Evaluation criteria:
Equivocal results should be clarified by further testing using modification of experimental
conditions.
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; twosided
test at P 0.05) increase in the frequency of rnicronucleated polychromatic
erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in
the data for male or fernale groups separately.
A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P
0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the
combined data for both sexes nor in the data for male or female groups separately.
The preceding criteria are not absolute and other modifying factors may enter into the final
evaluation decision.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic
erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by
counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only
counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that this test is valid and that Kn 172 is not mutagenic in the micronucleus test
under the experimental conditions described in this report.
Executive summary:

Micronucleus test in bone marrow cells of the mouse with Kn 172.

Kn 172 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on

erythrocytes in bone marrow.

The study procedures described in this report were based on the most recent OECD and EEC

guidelines.

Batch Kn-Gi 863411 of Kn 172 was a white powder with a purity of >99%. The test substance

was dissolved in physiological saline.

Five male and five fernale anirnals were used in each of the six treatment groups, including

negative and positive controls. All groups received a single intraperitoneal injection. The

negative and positive control groups were treated with vehicle and 50 rnglkg body weight of

cyclophosphamide (CP), respectively. Animals were dosed with Kn 172 at 30 (two groups), 15

(one group), and 7.5 (one group) mglkg body weight. Additional animals were dosed with the

highest dose level to compensate for possible deaths. After dosing the animals of the dose level

of 30 mglkg body weight showed the following toxic signs: lethargy, hunched posture, rough

coat (1 7 male anirnals and 12 female anirnals) and ptosis (4 male animals and one female

animal). Animals dosed with 15 mg Kn 172lkg body weight showed the following toxic signs:

rough coat (all male animals) and hunched posture (all male and 3 female animals). The

animals of the dose level of 7.5 rnglkg body weight showed no abnorrnalities after dosing.

Bone rnarrow of the groups treated with Kn 172 was sampled 24 or 48 (highest dose only) hours

after dosing. Bone rnarrow of the negative and positive control groups was harvested 24 and 48

hours after dosing, respectively.

Additional animals (three animals per time point) were dosed with 30 mg Kn 1721kg body weight

or with vehicle for blood sampling. Blood was sampled one and four hours after dosing of the

animals of the highest dose group. Blood from the vehicle control animals was sampled one

hour after dosing.

No increase in the mean frequency of rnicronucleated polychromatic erythrocytes was observed

in the polychrornatic erythrocytes of the bone rnarrow of anirnals treated with Kn 172.

The incidence of micronucleated polychromatic erythrocytes in the bone rnarrow of all negative

control animals was within the historical solvent control data range. Cyclophosphamide, the

positive control substance, induced a statistically significant increase in the number of

micronucleated polychromatic erythrocytes in both Sexes. Hence, both criteria for an acceptable

assay were met.

The groups that were treated with Kn 172 showed no decrease in the ratio of polychrornatic to

normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic

effects of this cornpound on the erythropoiesis. The groups that were treated with

cyclophosphamide showed an expected decrease in the ratio of polychromatic to

normochromatic erythrocytes cornpared to the vehicle controls, demonstrating toxic effects on

erythropoiesis.