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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:
13.04. - 19.05.2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Test concentrations with justification for top dose:
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as part of experiment I since the criteria mentioned above were met. Based on the toxic effects observed in the pre-experiment, seven concentrations were tested in experiment I and II. The confirmatory experiment I A was performed with six concentrations.
The concentration range included two logarithmic decades. The following concentrations were tested:
Experiment 1: 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
Experiment I A: 25; 50; 100; 150; 200; and 300 µg/plate
Experiment II (without S9 mix): Experiment II (with S9 mix): 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
Experiment II (with S9 mix): 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
deionised water
Controls
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1537, TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: yes, obeersered in all strains used
Remarks:
strong toxic effects, evident as a reduction in the number of revertants

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item induced gene mutations by frameshifts in the genome of the strains TA 1537 and TA 98 in the absence of metabolic activation.
Therefore, A 130 is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of A 130 to induce gene mutations according to the plate incorporation test (experiment I and I A) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in three independent experiments. Experiments I and II were performed with and without metabolic activation. Due to a questionable increase in revertant colonies in strain TA 100 in experiment I a confirmatory experiment was performed without metabolic activation. This confirmatory experiment is reported as experiment I A. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment 1: 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

Experiment I A: 25; 50; 100; 150; 200; and 300 µg/plate

Experiment II (without S9 mix): Experiment II (with S9 mix): 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

Experiment II (with S9 mix): 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Reduced background growth was observed in experiment I in strains TA 98 and TA 100 at 333 µg/plate and above and in strains TA 1535 and TA 102 at 1000 µg/plate and above in the absence of metabolic activation. In experiment I A reduced background growth was observed at 200 µg/plate and above. In experiment II in all strains reduced background growth was observed at 333 µg/plate and above without S9 mix and at 1000 µg/plate and above with S9 mix.

Strong toxic effects, evident as a reduction in the number of revertants, were observed in all strains used.

Substantial and dose dependent increases in revertant colony were observed following treatment with A 130 in strains TA 1537 (experiment I and II, without S9 mix) and TA 98 (experiment II, without S9 mix). The number of colonies exceeded the threshold of twice (strain TA 98) at 100 µg/plate and thrice (strain TA 1537) at 33 and 100 µg/plate the number of the corresponding solvent control. At higher concentrations the number of colonies was reduced due to overlapping toxic effects.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.