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Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-01-13 to 2012-02-17
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
according to guideline
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
according to guideline
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay

Test material


Target gene:
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
experiment I: rat S9 liver microsomal fraction; experiment II: hamster S9 liver microsomal fraction
Test concentrations with justification for top dose:
Experiment I and II:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
A third (confirmatory) experiment was performed with tester strain TA 100 (without metabolic activation) with the following concentrations:
31.6, 100, 316, 1000, 1750, 2500, 3750 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A.dest.
Untreated negative controls:
A. dest., BSL Lot No. 111219, 120105, 120203, 120305
Negative solvent / vehicle controls:
A. dest., BSL Lot No. 111219, 120105, 120203, 120305
Positive controls:
Positive control substance:
other: see remarks
Without metabolic activation: sodium azide (tester strains TA100, TA1535); 4-nitro-o-phenylene-diamine (TA98, TA1537); methylmethanesulfonate (TA102). With metabolic activation: 2-aminoanthracene for all strains; exception: TA 98, hamster liver: congored
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

- Preincubation period: 30 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 30 min at 30 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used (in a one case only two plates were evaluated).

- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
experiment II, TA 102
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Any other information on results incl. tables

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I, II and III with one exception: in experiment II in tester strain TA 102 a toxic effect was observed at concentrations of 2500 µg/plate and higher with metabolic activation.

No biologically relevant increases in revertant colony numbers were noted in tester strains TA 98, TA 1535, TA 1537 and TA 102 (with and without metabolic activation) and in tester strain TA 100 with metabolic activation.

An increase of revertant colony numbers was observed in tester strain TA 100 at concentrations of 2500 µg/plate and higher (without metabolic activation, only experiment I). A maximum mutation factor of 2.0 was reached at a concentration of 2500 µg/plate (without metabolic activation). A dose-response relationship was found.

In the confirmatory experiment (experiment III) these findings could not be verified. All colony number were within the historical data range, no increased mutation factors have been observed. Therefore, the increase of revertant colony numbers in experiment I was considered to be not biologically relevant.

Applicant's summary and conclusion

Interpretation of results (migrated information):

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, FAT 40858/A TE did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, FAT 40858/A TE is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria - modified for azo compounds-, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to FAT 40858/A TE at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I and II) and 31.6, 100, 316, 1000, 1750, 2500, 3750 and 5000 µg/plate (experiment III), in the presence and absence of mammalian metabolic activation according to the plate incorporation method with rat liver S9 (experiment I and III) and the pre-incubation method with hamster liver S9 (experiment II).

FAT 40858/A TE was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.