Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Nov to Dec 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd/Win: NMRI
- Source: Harlan Winkelmann GmbH (Borchen, Germany)
- Age at study initiation: approx. 6-12 weeks
- Weight at study initiation: 37-44 g
- Assigned to test groups randomly: yes
- Housing: individual in type I cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-23.0
- Humidity (%): 39-43
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
0.5 % aqueous Cremophor
Frequency of treatment:
twice, approximately 24 hours apart
Post exposure period:
no
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 250, 500 mg/kg bw
Basis:
other: actual injected
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (20 mg/kg bw) : dissolved in deionized water, single i.p. injection with 10 ml/kg bw

Examinations

Tissues and cell types examined:
The bone marrow was prepared by centrifugation. The numbers of polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) with and without micronuclei (MN) were counted.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The selection of the BAY 54-9085 doses was based on a pilot test, with groups consisting each of three males and three females. They received two intraperitoneally injections separated by 24 hours. The following doses were used: 500 mg/kg and 1000 mg/kg BAY 54-9085. In males and females the following symptoms were recorded for up to at least 48 hours after the second application, starting at 500 mg/kg: apathy, roughened fur, hard abdomen, spasm, difficulty in breathing and lachrymation. 3 of 3 males and 1 of 3 females died in the 1000 mg/kg group. In addition, 1 of 3 males died in the 500 mg/kg group.

Based on these findings, 500 mg/kg BAY 54-9085 were chosen as MTD for males. Due to the results of the dose range finder it is concluded, that there are no substantial differences between sexes in toxicity. Therefore, no females were used.


DETAILS OF SLIDE PREPARATION:
Schmid's method was used to produce the smears (Mutation Res. 31, 9-15, 1975).

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus. Normally, 2000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. In addition, the number of normochromatic erythrocytes per 2000 polychromatic ones was noted. If the ratio for a single animal amounts to distinctly more than 6000 normochromatic erythrocytes per 2000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omit-ted from the evaluation. A relevant, treatment-related alteration of the ratio polychromatic to normochromatic erythrocytes can only be concluded if it is clearly lower for a majority of the animals in the treated group than in the negative control.
Evaluation criteria:
A test was considered positive if there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of historical negative controls. In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. A test was also considered equivocal, if its result was implausible.
Statistics:
The BAY 54-9085 group(s) with the highest mean per time point (provided this exceeded the respective negative control mean) and the positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5 % and the treatment group figure was higher than that of the negative control. The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi2-test. A variation was considered statistically significant if the error probability was below 5 % and the treatment group figure was higher than that of the negative control.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
see "Additional information on results"
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
After two intraperitoneal administrations of 125, 250 and 500 mg/kg BAY 54-9085, treated males showed the following compound-related symptoms until sacrifice: apathy, roughened fur, hard abdomen, spasm, periodically stretching of body and difficulty in breathing. There was no substance-induced mortality. No symptoms were recorded for the control groups. No animals died in these groups.

There was no altered ratio between polychromatic and normochromatic erythrocytes.

After two intraperitoneal treatments of males with doses up to and including 500 mg/kg no indications of a clastogenic effect of BAY 54-9085 were found.

Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.

Any other information on results incl. tables

Table 1: Summary of results from micronucleus test with BAY 54-9085

 Treatment group

 Dose i.p. (mg/kg bw)

 Number of evaluated PCE

 Number of NCE per 2000 PCE

 MNNCE per 2000 NCE

 MNPCE per 2000 PCE

 Negative control

0

10,000

1873± 427

3.2 ± 1.4

2.4 ± 1.1

 

 

 

 

 

 

 BAY 54-9085

2 x 125

10,000

1017 ± 333

1.6 ± 1.5

2.8 ± 1.8

 

2 x 250

10,000

 902 ± 148

2.3 ± 0.4

2.4 ± 0.9

 

2 x 500

10,000

1039 ± 304

0.8 ± 1.1

3.2 ± 1.5

 

 

 

 

 

 

 Positive control

 (Cyclophosphamide)

1 x 20

10,000

1638 ± 498

2.3 ± 2.5

32.2* ± 12.2

* p<0.01 in non-parametric Wilcoxon ranking test

 

NCE = normochromatic erythrocytes

PCE = polychromatic erythrocytes

MNNCE = micronucleated NCE

MNPCE = micronucleated PCE

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

Male mice treated with BAY 54-9085 received two intraperitoneal administrations of 125, 250 and 500 mg/kg bw, respectively, separated by 24 hours. Males of the positive control received a single intraperitoneal treatment with 20 mg/kg cyclophosphamide. The femoral marrow of all groups was prepared, 24 hours after the last administration. Males treated twice with BAY 54-9085 in doses up to 500 mg/kg showed symptoms of toxicity after administration, starting at 125 mg/kg. However, all males survived until the end of the test. There was no altered ratio between polychromatic and normochromatic erythrocytes. After two intraperitoneal treatments of males with doses up to and including 500 mg/kg no indications of a clastogenic effect of BAY 54-9085 were found.

 

Thus, BAY 54-9085 was concluded to be negative in the mouse micronucleus assay.