4-mesyl-2-nitrotoluene

Genetic toxicity: in vitro

Administrative data

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:

from 2012-01-16 to 2012-02-02 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-550-0

EC Name:

-

Cas Number:

1671-49-4

Molecular formula:

C8H9NO4S

IUPAC Name:

4-methanesulfonyl-1-methyl-2-nitrobenzene

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical state: White powder
- Stability under test conditions: not reported
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Histidine for Salmonella
Tryptophan for E. Coli

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli, other: WP2 uvrA pKM101, WP2 pKM101

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fraction

Test concentrations with justification for top dose:

Experiment I, without S9: 50, 150, 500, 1500, 5000 µg/plate
Experiment I, with S9: 50, 150, 500, 1500, 5000 µg/plate, 5 and 15 µg/plate in addition for strain TA1537 and WP2uvrA pKM101
Experiment II, without S9: 5, 15, 50, 150, 500, 1500, 5000 µg/plate, 1.5 µg/plate in addition for strain WP2uvrA pKM101 and WP2pKM1001
Experiment II, with S9: 5, 15, 50, 150, 500, 1500, 5000 µg/plate, 1.5 µg/plate in addition for strain WP2uvrA pKM101 and WP2pKM100

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was fully soluble in dimethyl sulphoxide at 50 mg/mL . The choice of solvent vehicle was based on its ability to solubilise the test item and lack of toxicity to the bacterial systems.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I with and without S9, Experiment II without S9: in agar (plate incorporation)
Experiment II with S9: preincubation

DURATION
- Preincubation period: 60 min (Experiment II with S9)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Toxic effects were evident as a reduction in the growth of the bacterial background lawns and/or number of revertants.

Evaluation criteria:

The test item will generally be considered mutagenic to bacteria if the following criteria are achieved. If the following criteria are not achieved then the test item will be considered non-mutagenic to bacteria.

i) In all strains a two-fold increase in the mean number of revertants per plate compared to the mean value of the concurrent vehicle control.
ii) Increases in revertant numbers for all strains must be related to increases in test item concentration.
iii) A positive response in one tester strain either with or without exogenous metabolic activation is sufficient to designate the test item as a bacterial mutagen.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test item activity. Results of this type will be reported as equivocal.

Statistics:

not performed

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Lowered revertant counts (less than 0.5 fold compared to the concurrent vehicle controls) were noted at 5000 µg/plate to Salmonella strain TA1537 in the presence of S9-mix (plate incorporation methodology).

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli, other: WP2 uvrA pKM101, WP2 pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Lowered revertant counts (less than 0.5 fold compared to the concurrent vehicle controls) were noted at 5000 µg/plate to Escherichia coli strains WP2uvrApKM101 and WP2pKM101 in both the presence and absence of S9-mix.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: Not performed

COMPARISON WITH HISTORICAL CONTROL DATA: THe number of revertants of the solvent control and the positive control lie within the hitsorical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test item caused no visible reduction in the growth of the bacterial background lawns of any of the tester strains in either the absence or presence of S9-mix. However, lowered revertant counts (less than 0.5 fold compared to the concurrent vehicle controls) were noted at 5000 µg/plate to Escherichia coli strains WP2uvrApKM101 and WP2pKM101 in both the presence and absence of S9-mix (plate incorporation and pre-incubation methodology) and to Salmonella strain TA1537 in the presence of S9-mix ( plate incorporation methodology). The test item was tested up to the maximum recommended dose level.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

This GLP-study was performed to investigate the potential of the test item to induce gene mutation..The method followed was that outlined in OECD TG 471.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strains WP2uvrApKM101 and WP2pKM101 were treated with the test item using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (performed using the plate incorporation method) ranged between 5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. A second experiment (utilising the pre-incubation method dosed in the presence of S9 and plate incorporation dosed in the absence of S9) was performed at later dates using doses ranging from 1.5 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test item formulations. Additional dose levels and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. The positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 The test item caused no visible reduction in the growth of the bacterial background lawns of any of the tester strains in either the absence or presence of S9-mix. However, lowered revertant counts (less than 0.5 fold compared to the concurrent vehicle controls) were noted at 5000 µg/plate to Escherichia coli strains WP2uvrApKM101 and WP2pKM101 in both the presence and absence of S9-mix (plate incorporation and pre-incubation methodology) and to Salmonella strain TA1537 in the presence of S9-mix ( plate incorporation methodology). The test item was tested up to the maximum recommended dose level. No test item precipitate was observed on the plates at any of the dose levels tested in either the presence or absence of S9-mix.

 No significant increases in the frequency of revertant colonies, in excess of twofold greater than the concurrent solvent controls, were recorded for any of the bacterial strains, for any dose level of the test item, either with or without metabolic activation.

The test item was considered to be non-mutagenic under the conditions of this test.