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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study mainly followed the principles laid down in the OECD Guideline for Testing of Chemicals No. 471.

Data source

Reference
Reference Type:
publication
Title:
Mutagenicity of carbon tetrachloride and chloroform in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, using a gas exposure method.
Author:
Araki A, Kamigaito N, Sasaki T, Matsushima T
Year:
2004
Bibliographic source:
Environmental and Molecular Mutagenesis, 43(2), 128-133.
Report Date:
2004

Materials and methods

Principles of method if other than guideline:
Gas phase exposure using a gas sampling bag (Araki, A., Noguchi, T., Kato, F., and Matsushima, T. 1994. Mutat. Res. 307, 335-344.)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance: chloroform
CAS no.: 67-66-3
Chloroform contained 0.3-1.0% of ethanol as a stabilizer.
Source: Wako Pure Chemical Industries
Batch: APJ7886
Purity: 99.7%

Additional source: Tokyo Kasei Kogyo
Batch: GG01
Purity: >99.0%

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix (4mM NADPH, 4mM NADH, 5mM G-6-P, 8mM MgCl2, 33mM KCl, 100mM sodium phosphate buffer pH 7.4, 10% S9); S9 Mix with additional glutathione (10mM GSH)
Test concentrations with justification for top dose:
0.01, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 % in the gas phase
Vehicle / solvent:
Vehicle/solvent: none
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
other: air only
True negative controls:
no
Positive controls:
no
Positive control substance:
no
Remarks:
no
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
no
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2furyl)acrylamide
Remarks:
no
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
no
Details on test system and experimental conditions:
Gas-phase exposure using a gas sampling bag [Araki et al., 1994] was conducted with slight modifications. A (l.5 ml. aliquot of S9* mix (for assays with metabolic activation) or 0.1 M sodium phosphate buffer (pH 7.4: for assays without metabolic activation) plus 0.1 mL of tester strain. which had been cultured at 37°C for 10 h in nutrient broth, were mixed with 2 mL of molten top agar containing 0.05 µmol/ml of L-histidine and D-biotin (for tests conducted with Salmonella) or 0.05 µmol/ml_ of L-tryptophan (for tests conducted with E. coli), and immediately poured into plates containing 30 mL of Vogel-Bonner minimal glucose agar (Oriental Yeast). The plates were kept on a flat table until the top agar solidified. The plates then were placed separately, upside down without their lids, in a plate holder, arranging the plates according to their level of exposure and the activation system used. The plate holder was placed in a 10L gas exposure bags through an opening made by scissors in one side of the bag. The bag was then closed by folding the open side 3 times and sealing with adhesive tape. The exposure concentrations used in the test were calculated from the volumes of the test compound vapor and air diluent in the gas exposure bag at the exposure temperature. First, the air trapped in the exposure bag after loading the plate holder was removed using a pump. A fixed volume of diluent air to produce a final exposure volume of 500 mL per plate then was pumped into the gas exposure bag. The test compound was injected through a septum directly into the gas exposure bag using a syringe. and the solution in the bag was vaporized by placing the portion of the bag making contact with the test agent on a hot-plate (at 50-60°C). After vaporization, the bag was squeezed by hand, to mix the test compound vapor and air thoroughly. The bacterial plates in the gas exposure bag were incubated at 37°C for 24 h. After the 24 h exposure, the test compound vapor was removed from the gas exposure bag. The seal on the gas exposure bag was peeled off, and the plate holder was taken out of the bag. The plates were held in a safety cabinet for 10 min in order to remove the test compound, and then the lids of the plate were replaced. The plates with lids were turned upside down. transferred separately to a vinyl bag, according to their concentration level and whether with/without the S9 mix, and incubated an additional 24 h at 37°C. The maximum exposure concentration was 5%. Chloroform showed toxicity for all strains in tests conducted with 5%. The sensitivity of tester strains and the activity of the S9 mix were established by testing positive control substances by the pourplate method. Two plates per dose were employed in assays conducted with the test agents: negative control assays (exposure to airs used 4 plates. *S9 was a liver homogenate fraction prepared from a male Sprague-Dawley rat pretreated with sodium phenobarbital and 5,6-benzoflavone.
Evaluation criteria:
The twofold rule (Ames, B.N., McCann, J., and Yamasaki, E. 1975. Mutat. Res. 31, 347-364) was used for determining mutagenicity in individual experiments.
Statistics:
To compare the response for a particular dose group in a particular tester strain (in some cases more than three data values per dose group from different experiments) with those of the concurrent control group, Bartlett's test was first used to determine whether the variance was homogeneous or not. If the variance was homogeneous, one-way ANOVA was applied. Else, the Kruskal-Wallis rank sum test was performed, arranging all data for control and dose groups in descending order. Statistical differences in means and rank means among the groups then were analyzed by Dunnett's multiple comparison test, and the same multiple comparison test by rank, respectively. Two-sided analyses were performed, with the p-values for significance set at 0.05 and 0.01.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 5.0%
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Chloroform was not mutagenic in Salmonella typhimurium strains TA98, TA 100, TA 1535, or TA 1537, with or without S9 mix, and no mutagenicity was observed in TA98, TA 100, TA 1535, TA 1537 in the presence of glutathione-supplemented S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative TA1535, TA1537, TA98, TA100

Exposure of Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 to chloroform via the gas phase did not cause mutagenicity up to a dose of 2 %.
Executive summary:

The present study investigated the mutagenicity of chloroform in Salmonella typhimurium strains TA98, TA100, TA1535, and TA 1537 using a gas exposure method. Mutagenicity was tested with and without a metabolic activation system and additionally with a glutathione-supplemented metabolic activation system.

The tester strains were exposed for 24 hours to chloroform concentrations in the gas phase of 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2 and 5 % at 37°C. The sensitivity of tester strains and the activity of the S9 mix were established by testing positive control substances by the pour-plate method. Two plates per dose were employed in assays conducted with the test agents. Negative control assays (exposure to air) used four plates.

The maximum exposure concentration of 5 % chloroform in the gas phase was toxic for all strains. Chloroform was not mutagenic in TA98, TA100, TA1535 or TA1537, with or without S9 mix, and no mutagenicity was observed in TA98, TA100, TA1535 or TA1537 in the presence of glutathione-supplemented S9 mix.