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Toxicological information

Genetic toxicity: in vivo

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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:
1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to valid testing and GLP guidelines, and was considered relevant, adequate and reliable for classification purpose.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report Date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Chloracetamide
- Physical state: White crystalline powder
- Analytical purity: Technically pure
- Other: Manufactured by Hoechst AG (Gersthofen) and handed over together with study order dd 1982-03-24; Test solutions were freshly prepared

Test animals

Species:
mouse
Strain:
other: NMRI HOE NMRKf (SPF71)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
-Source: Own breeding (Pharma Forschung Toxikologie, Kastengrund, Hattersheim, Germany)
-Weight : Before first administration males 32-42 g (average 37 g) and females 24-30 g (average 28 g)
-Age: 7-12 weeks
-Housing: Until 5 animals of the same sex per cage with mesh cover (Type 3) on softwood pellets
-Diet: Altromin 1324 (Altromin GmbH, Lage/Lippe (Germany)), ad libitum
-Water: Tap water in plastic drinkers, ad libitum

ENVIRONMENTAL CONDITIONS
-Temperature: 23-26°C
-Humidity: 42-49%

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
desalinated water
Details on exposure:
2x oral dose, 2nd dose given after 24 h
Duration of treatment / exposure:
total study period: 30 h
Frequency of treatment:
2 times
Post exposure period:
1st application: 24 h
2nd application: 6 h
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1, 10, 100 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Positive control(s):
5 males / 5 females, 2x 100 mg Cyclophosphamid / kg bw
same application periods as for the test substance

Examinations

Tissues and cell types examined:
Bone marrow: erythrocytes, micronucleus containing cells
Details of tissue and slide preparation:
Preparation of the femurs, use gauze to remove muscle tissues from the bone.
Opening of the proximal end, flushing out the bone marrow using 1 mL of fetal calf serum (FCS).
Dilute with FCS and centifuge at 1000/ min, remove supernatant.
Equilibrate sediment & apply 1 drop unto the plate.
Dry 24 h, dye with may-Gruenwalds-solution, wash, dye with Giemsa solution, wash & dry.
Embed in Entellan TM (Merck, Germany)
Evaluation criteria:
No. of polychromatocytes/Normocytes
Micronucleus containing cells / 2000 polychromatic erythroctes
Micronucleus containing normocytes / 1000 normocytes
Statistics:
Procedure acc. to Nemenyi; 95 % significance level.The number of micronucleated polychromatic erythrocytes/2000 counted polychromatic erythrocytes and the number of micronucleated normocytes / 1000 counted normocytes was statistically analysed. The binominal distribution was used to examine the increase of micronucleated cells compared to the simultaneous controls.
In addition, the ratio of polychromatic to normochromatic erythrocytes was evaluated statistically. Any differences compared to the simultaneous controls separated by gender were tested by the method of Nemenyi.
Statistacal analysis was perfomed by a computer program, created by the department for practical mathematics of Hoechst AG.
All the statistical results are based on the 95% significance level.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Following all doses of Chloroacetamide, rates of micronucleus-containing polychromatic erythrocytes as well as rates of normocytes were in normally ranges of spontaneous formations and were not significantly different from the values of the control animals.

Ratio of polychromatic erythrocytes vs normocytes were not influenced by chloroacetamide.

Cyclophosphamide induced a markedly increase of micronucleus containing polychromatic erythrocytes.
The increase was statistically significant for both sexes.
Further, the ratio of polychromatic erythrocytes vs normocytes became shifted to the matured cells.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Study results do not indicate chromosome aberrations by chloroacetamide.
Executive summary:

Chloroacetamide was administered to mice by oral gavage twice at an interval of 24 h at doses of 0, 1, 10, 100 mg/kg bw, next to a positive control (Cyclophophamide 100 mg/kg bw).Six hours after the second administration, the animals were sacrificed and the bone marrow obtained from the femurs was processed, smeared on slides and stained. Thereafter, normocytes and polychromatic erythrocytes of each animal were examined for the ratio immature cells / normocytes as well as the number of micronucleated normocytes and polychromatocytes. Chloroacetamide did not result in a significant increase in the number of polychromatic erythrocytes with micronuclei under the conditions of the experiment; the number of micronucleated normocytes also did not show substance related changes. The ratio normocytes/polychromatic erythrocytes in male and female animals in all dose groups was unaffected. The present result do not indicate an induction of chromosomal mutations by Chloroacetamide.

Cyclophosphamide produced a significant increase in the number of micronucleated polychromatic erythrocytes in male and female animals. As an expression of the cytostatic effect of Cyclophosphamide also the ratio normocytes/polychromatic erythrocytes was shifted in favor of the mature cells.