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Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
CAS No.: 78567-28-9; Batch No.: WDJ 1853 D-3; Appearance: clear colorless viscous liquid
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:
Following doses of the test substance were evaluated: 5000; 1600; 500; 160; 50; 16 ug/plate.
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
mitomycin C
other: nitrofurantoin, 4-nitro-1,2-phenyiene diamine, cumene hydroperoxide and 2-aminoanthracene
Details on test system and experimental conditions:
The initial plate incorporation method:
For the mutant count, one plate was used, both with and without S9 mix for each strain and dose. One plate filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained one plate per station. In experiments without S9 mix buffer was used as replacement.
The amount of solvent for the test substance and for the controls was 0.1 ml/plate.
The doses for the first trial were routinely determined on the basis of the standard protocol: if not limited by solubility 5000 ug or 5 uL per plate were used as the highest dose. At least five additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed.
The independent repeat was performed as reintubation in a water bath at 37 °C for 20 min. At the end of the reintubation period 2 ml of molten soft agar were added to the tubes, the content mixed and plated.
For the mutant count, one plate was used for each strain and dose. One plate filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained one plate per station. In experiments without S9 mix buffer was used as replacement.
The doses of this trail were determined on the basis of the results of the plate incorporation assay.
The toxicity of the substance was assessed in three ways:
- Gross appraisal of background growth on the plates for mutant determination.
- Marked and dose dependent reduction in the mutant count per plate compared to the negative controls.
- Titer was determined. The dilution of bacterial suspensions used for the determination of titers was 1:1000000. Titers were determined under the same conditions as were mutations, except that the histidine concentration in the soft agar was increased five-fold to permit the complete growth of bacteria.
The tests were performed with and without S9 mix. The counts were made after plates were incubated for 48 h at 37 °C.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>160 ug/plate
Untreated negative controls validity:
valid
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
Remarks:
>160 ug/plate
Untreated negative controls validity:
valid
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
Remarks:
>160 ug/plate
Untreated negative controls validity:
valid
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
Remarks:
>160 ug/plate
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>160 ug/plate
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The Ames test screening employing doses up to 5000 ug per plate showed the test substance to produce bacteriotoxic effects starting at 160 ug/plate.
None of the five strains concerned showed in the plate incorporation test a dose-related and biologically relevant increase in mutant counts over those of negative controls (with and without S9 mix).
The positive controls increased mutant counts to well over those of the negative controls, and thus demonstrated the system’s sensitivity and the activity of the S9 mix.
Conclusions:
Under the study conditions, the test substance was not considered to be mutagenic in Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the absence and presence of metabolic activation.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B.13/14. The test substance was evaluated using five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). Following concentrations of the test substance were evaluated: 0, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following incubation in the plate incorporation assay, the test substance produced cytotoxic effects starting at 160 ug/plate. None of the five strains tested showed a dose-related and biologically relevant increase in mutant colony counts over those of negative controls (with and without S9 mix). Under the study conditions, the test substance was not considered to be mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the absence and presence of metabolic activation (Herbold, 2002).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B.13/14. The test substance was evaluated using five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). Following concentrations of the test substance were evaluated: 0, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following incubation in the plate incorporation assay, the test substance produced cytotoxic effects starting at 160 ug/plate. None of the five strains tested showed a dose-related and biologically relevant increase in mutant colony counts over those of negative controls (with and without S9 mix). Under the study conditions, the test substance was not considered to be mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the absence and presence of metabolic activation (Herbold, 2002).

Justification for classification or non-classification