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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The information is reliable and adequate for covering the endpoint.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
liquid

Method

Target gene:
S. typhimurium TA1535 hisG46 rfa uvrB; S. typhimurium TA1537 hisC3076 rfa uvrB; S. typhimurium TA 1538 hisD3052 rfa uvrB; S. typhimurium TA98 hisD3052 rfa uvrB pKMl01; S. typhimurium TA100 hisG46 rfa uvrB pKM101; E. coli WP2 uvrA trp
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rats (S9 mix)
Test concentrations with justification for top dose:
Exp 1: with and without S9-mix, 10 dose levels between 9.8 and 5000 µg/plate
Exp 2 with and without S9-mix
for TA1535, TA1537 and WP2 uvrA: 6 dose levels between 39.1 and 12500 µg/plate
for TA1538, TA98 and TA100: 8 dose levels between 39.1 and 12500 µg/plate
Vehicle / solvent:
dimethyl sulphoxide (DMSO)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
and 9-Aminoacridine, 2-Nitrofluorene and 2-Aminoanthracene
Details on test system and experimental conditions:
An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 100 µl of the test solution was added, followed immediately by 2 ml of histidine/tryptophan deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Each petri dish was individually labelled with a unique code corresponding to a sheet, identifying the dish's contents. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, 59 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period the appearance of the background bacterial lawn was examined and revertant colonies counted using a Seescan Automatic Colony Counter.
Evaluation criteria:
Please see below.
Statistics:
no

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity was observed towards all the strains at the top three dose levels, down to 625 µg/plate for TA1538 and down to 312.5 µg/plate for TA98 and TA100. In the second mutation test toxicity was observed towards all the strains at the top dose level and down to 312.5 µg/plate for TA1538, TA98 and TA100.
No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment at any dose level, in the presence or absence of S9 mix, in either mutation test.

Applicant's summary and conclusion

Conclusions:
It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in the absence and presence of the S9-mix, indicate that the test substance is not mutagenic under the conditions of this test.
Executive summary:

In a study, performed in accordance with GLP and OECD guideline 471 and 472, the test substance was examined for its possible mutagenic activity in the bacterial reverse mutation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and the Escherichia coli strain WP2uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix).The test substance was diluted in dimethylsulphoxide (DMSO). Two independent mutation tests were performed. A top dose level of 5000 µg/plate, based on a preliminary test, was chosen for the first mutation study. Other dose levels used were a series of 2-fold dilutions of the highest concentration. A total of ten dose levels were used in the first test to ensure that sufficient non-toxic concentrations were assessed. Toxicity was observed towards all the strains at the top three dose levels, down to 625 µg/plate for TA1538 and down to 312.5 µg/plate for TA98 and TA100. For the second mutation test a top dose level of 12500 µg/plate was chosen and the bottom two dose levels not included for TA1535, TA1537 and WP2 uvrA. Toxicity was observed towards all the strains at the top dose level and down to 312.5 µg/plate for TA1538, TA98 and TA100. No evidence of mutagenic activity was seen at any dose level in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that the test substance shows no evidence of mutagenic activity in this bacterial system.