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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Bacterial reverse mutation assay: negative; OECD 471, Donath 2012
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to guideline; under GLP conditions.
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media: Stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (8% w/w) )over liquid nitrogen.
- All strains contain mutations in the histidine operon. They contain the deep rough (rfa) mutation and deletion of the uvrB gene. Strains TA98, TA100 and TA102 contain the R-factor plasmid, pkM101.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction from rats treated with phenobarbital/beta-napthoflavone
Test concentrations with justification for top dose:
Preliminary test concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 ug/plate
Main test: 31.6, 100, 316, 1000, 2500 and 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the vehicle based on solubility of the test material and compatibility with the target cells.
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: sodium azide (NaN3); 4-nitro-o-phenylene-diamine (4-NOPD); methylmethanesulfonate (MMS). With metabolic activation: 2-aminoanthracene (2-AA).
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 - in agar (plate incorporation); Experiment 2 - preincubation.

DURATION
- Preincubation period: 60 minutes (experiment 2 only).
- Expression time (cells in growth medium): Plates were incubated at 37 degrees C for at least 48 hours

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used as a minimum.

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity was determined by a clearing or diminuation 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.

OTHER: PRE-EXPERIMENT FOR TOXICITY: To evaluate the toxicity of the test article, a pre-experiment was performed with strains TA98 and TA100. Eight concentrations (3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 ug/plate) were tested for toxicity and mutation induction with each 3 plates.
Evaluation criteria:
Criteria of validity: A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical response to ampicillin (TA98, TA100, TA102)
- the control plates with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range): TA98: without S9: 16-46, with S9: 18-56; TA100: without S9: 77-174, with S9: 80-165; TA1535: without S9: 5-29, with S9: 5-27; TA1537: without S9: 5-28, with S9: 5-34; TA102: without S9: 164-399, with S9: 163-458.
- Corresponding background growth on negative control, solvent control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.

Evaluation of mutagenicity: 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 TA98, TA100 and TA102 the number of reversions is at least twice as high as the reversion rate of the solvent control.
- If in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In a preliminary toxicity assay, the maximum dose tested was 5000 ug/plate. Based on the findings of the preliminary toxicity assay, the maximum doses tested in the mutagenicity assay 5000 ug/plate in the presence and absence of S9 with all tester strains.

No precipitation of the test material was observed in any tester strains evaluated in experiments 1 and 2.

Toxic effects of the test material were noted in several tester strains evaluated in experiments 1 and 2. In experiment 1, toxic effects of the test item were observed in strain TA1535 at a concnetration of 5000 ug/plate (without metabolic activation) and at concentrations of 2500 ug/plate and higher (with metabolic activation). In experiment 2, toxic effects of the test material were noted in strains TA100 and TA1537 at a concentration of 5000 ug/plate (without metabolic activation). In strain TA1535, toxic effects of the test material were noted at concentrations of 2500 ug/plate and higher (without metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.4 found in experiment 2 in strain TA1537 at a concentration of 100 ug/plate (without metabolic activation) was regarded as not biologically relevant due to the lack of a dose-response relationship.

No biologically relevant increases in revertant colony numbers of any of the 5 tester strains were observed following treatment with the test material at any concentration level, neither in the presence nor absence of metabolic activation in experiments 1 and 2.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test material is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

The study was performed to evaluate the potential mutagenicity of the test material in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA102, in both the presence and absence of S9 mix. The study was performed to GLP and was designed to the requirements of OECD 471, EU Method B.13/14, EPA OPPTS 870.5100. In a preliminary toxicity assay, the maximum dose tested was 5000 ug/plate. Based on the findings of the preliminary toxicity assay, the maximum doses tested in the mutagenicity assay 5000 ug/plate in the presence and absence of S9 with all tester strains. Positive controls appropriate for each strain, in the presence and absence of S9 mix, were included.

Two independent experiments were performed with and without metabolic activation. The concentrations, including the controls were tested in triplicate. The test concentrations used in the main study were: 31.6, 100, 316, 1000, 2500 and 5000 ug/plate. The test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were clearly demonstrated by the increases in the numbers of reverant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 in both the presence and absence of S9 mix.

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

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity in vitro, ames test: The study was performed to evaluate the potential mutagenicity of the test material in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA102, in both the presence and absence of S9 mix. The study was performed to GLP and was designed to the requirements of OECD 471, EU Method B.13/14, EPA OPPTS 870.5100. In a preliminary toxicity assay, the maximum dose tested was 5000 ug/plate. Based on the findings of the preliminary toxicity assay, the maximum doses tested in the mutagenicity assay 5000 ug/plate in the presence and absence of S9 with all tester strains. Positive controls appropriate for each strain, in the presence and absence of S9 mix, were included.

Two independent experiments were performed with and without metabolic activation. The concentrations, including the controls were tested in triplicate. The test concentrations used in the main study were: 31.6, 100, 316, 1000, 2500 and 5000 ug/plate. The test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were clearly demonstrated by the increases in the numbers of reverant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 in both the presence and absence of S9 mix.


Justification for selection of genetic toxicity endpoint
One study available; GLP compliant and has a Klimisch score 1.

Justification for classification or non-classification

The substance does not meet classification criteria under EU Directive 67/548/EEC for mutagenicity.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity.