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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
7 in vitro and 3 in vivo genetic toxicity studies were performed. All the test results were negative except the chromosome aberration test in V79 lung cells of Chinese hamster in vitro and the Klebsiella gene mutation fluctuation test in vitro. These positive findings were overruled by the negative findings in vivo.
Link to relevant study records
Reference
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.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: NMRI HOE NMRKf (SPF71)
Sex:
male/female
Details on test animals or test system 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%
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
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
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.
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.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Both batteries of in vitro and in vivo genetic toxicity studies were performed.

In a key in vitro bacterial reverse mutation study (plate incorporation), Chloroacetamide solved in DMSO was tested with Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, with and without metabolic activation (S9) at concentrations of 4 to 2500 µg/plate (Hoechst, 1979). There was no mutagenicity neither in the presence nor absence of rat liver preparation up to 2500 µg/plate. Supporting information for absence of mutagenicity potential was available from an in vitro bacterial reverse mutation study (plate incorporation) with Konservierungsmittel CA 24 (containing 70% Chloroacetamide) with S.typhimurium strains TA 98, TA100, TA1535, TA1537 and TA1538 strains, with and without metabolic activation (S9) at concentrations of 0.5 to 1000 µg ( IBR, 1979) and from a published study with Chloroacetamide tested in S. typhimurium strains TA 98, TA 100 and TA 1537 with and without S-9 mix at concentrations ranging from 0.05 to 10 mg/plate (Voogd, 1989). A concentration of 20 mg/plate stopped bacterial growth. Finally, Chloroacetamide was also tested in a fluctuation test using K. pneumoniae at concentrations of 0.1 to 2 g/L (Voogd et al., 1989), showing some increased rate of mutation at and above 0.5 g/L and demonstrating cytotoxicity at 2 g/L.

In a key in vitro chromosome aberration study, Chloroacetamide was investigated in Chinese hamster lung V79 cells (Aventis, 2001) at 100, 250 and 350 µg/mL (3h with and without S9). The highest concentration produced distinct lowering (+- 50%) of the mitotic index, without precipitation of the test substance. A significant increase in the number of chromosome aberrations was induced at cytotoxic concentrations both with and without metabolic activation, however not below cytotoxic concentrations. In conclusion, Chloroacetamide was considered to be clastogenic at cytotoxic concentrations in this in vitro chromosome aberration assay with V79 Chinese hamster lung cells.

In a key in vitro mammalian gene mutation study (Aventis, 2001) Chloroacetamide was tested in HPRT V79 cells of Chinese hamster. Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix). In experiment 1, concentrations were 1 - 175 µg/mL without S9-mix and 5 - 225 µg/mL with S9 -mix; in the second main experiment, concentrations were 10 - 200 µg/mL without S9-mix and 50 - 250 µg/mL with S9 -mix. The highest concentrations showed toxic effects with and without metabolic activation. In both experiments in the absence of S9 metabolic activation no statistically significant increase in the mutant frequency was found with any of the concentrations used. Slight and sporadic statistically significant increases in the mutation frequency were found in the presence of S9, however they were not significant. Chloroacetamide did not induce gene mutations in this HPRT-test both in the presence and absence of a metabolic activation system and is considered to be non-mutagenic in this HPRT assay.

A supporting study was done with Chloroacetamide in an in vitro Cell Transformation Assay in Syrian Hamster Embryo (SHE) cells (CCR, 1987). Per test group 10 plates were exposed to the test article for 4h and 48h in the absence and 4h in the presence of S9-mix. Within 10 days after initiation of the cell culture the cells were fixed, stained and examined for morphological transformation. The test article did not significantly increase the number of morphologically transformed colonies up to cytotoxic concentrations in the absence of S9-mix for 4 and 48h, neither in the presence of S9-mix for 4h. Chloroacetamide did not induce morphological transformations, therefore it was concluded to have no transforming potential in this SHE cell transformation assay.

Additionally, in vivo studies were conducted. In a key bone marrow micronucleus assay, Chloroacetamide was administered twice to mice by oral gavage at an interval of 24 h at doses of 0, 1, 10, 100 mg/kg bw (Hoechst, 1982). 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 of 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 relevant changes. The ratio normocytes/polychromatic erythrocytes in male and female animals in all dose groups was unaffected. The present results do not indicate an induction of chromosomal mutations by Chloroacetamide. In a supporting study, Konservierungsmittel CA24 formulation (containing 70% Chloroacetamide) was tested for the influence on the chromosomes of the bone marrow and the testicular tissue in the Chinese hamsters, as well as induction of micronuclei in blood smears from bone marrow (IBR, 1979). Doses were given twice by intraperitoneal administration with 24h interval: 12.5, 25 and 50 mg/kg bw, corresponding with 1/12, 1/6 and 1/3 of the LD50 (= 150 mg/kg bw). Under the given experimental conditions, no increase in structural and numerical chromosome aberrations nor micronuclei were observed in treated groups.

Finally a supporting study was conducted by intraperitoneal administration of Konservierungsmittel CA 24 in a dominant lethal test at 114 mg/kg bw (group 1) and 123 mg/kg bw (group 2) in male NMRI-mice (IBR, 1979). The treated male mice were each mated with 3 virgin females per week over a period of 10 weeks. At autopsy of the mated females, a marked inhibitory effect on fertility of the treated males was observed, which was considered to be related to toxic effects in the male reproductive system during the first 3 weeks of testing. Since there were no changes in number of resorptions, mutagenicity index and number of dead fetuses during the entire test period, a mutagenic effect of the test substance under the given experimental conditions cannot be assumed.

In conclusion, based on the in vitro and in vivo test batteries, Chloroacetamide does not have a mutagenicity or chromosome aberration potential.


Justification for selection of genetic toxicity endpoint
This in vivo study for chromosome aberration was conducted according to valid testing and GLP guidelines, and was considered relevant, adequate and reliable for classification purpose in combination wiht the in vitro assays for bacterial & mammalian gene mutation.

Justification for classification or non-classification

Based on the findings in the genotoxicity test batteries, classification for genotoxicity is not warranted.