Reaction mass of mixed xylenes and sulphur monochloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17th July 2015 - 6th August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of Mixed Xylenes and Sulphur Monochloride

IUPAC Name:

Reaction Mass of Mixed Xylenes and Sulphur Monochloride

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 4600058
- Purity test date: Not applicable; substnace is a UVCB substance

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1 - Plate incorporation method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 micro grams/plate
Experiment 2 - Pre-incubation method: 15, 50, 150, 500, 1500 and 5000 micro grams/plate
The dose range for experiment 2 was determined from the results in experiment 1. Six dose concentrations were used to achieve four non-toxic dose levels and the potential toxic limit of the test item.

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1) and pre-incubation (Experiment 2)
DURATION
- Pre-incubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C for both application methods.
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells):N/A

NUMBER OF REPLICATIONS: 3 replicates at each dose

Evaluation criteria:

All tester strains should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
All tester strain cultures should be in the range 0.9 to 9x1^09 bacterial /ml.
Positive controls should be included to demonstrate the sensitivity of the tester strains and the integrity of the S9 mix. All positive controls should induce a marked increase in the frequency of revertant colonies with and without metabolic activation.
There should be a minimum of four non-toxic dose levels and no evidence of excessive contamination.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, no increases in the frequency of revertant colonies was observed, the test material 'reaction mass of mixed xylenes and sulphur monochloride' was considered to be non-mutagenic.

Executive summary:

The genetic toxicity of ‘reaction mass of mixed xylenes and sulphur monochloride was examined in a bacterial reverse mutation assay (Ames test) performed to GLP and in accordance with OECD 471 and EU method B.13/14. Salmonella Typhimurium strains TA1535, TA 1537, TA98 and TA100, along with Escherichia coli strain WP2uvrA, were tested with and without S9 rat liver fraction metabolic activation.

 Two mutation tests were performed, Experiment 1 used the plate incorporation method, test substance concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000µg/plate were assessed. Experiment 2 was performed using the pre-incubation method at concentrations of 15, 50, 150, 500, 1500 and 5000 μg/plate. Positive and vehicle controls were also tested.

There was no visible reduction in the growth of the background bacterial lawn at any dose level either with or without the metabolic activation, using either the plate incorporation method or the pre-incubation method. A test item precipitate was observed at the 5000 µg dose , this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of the revertant colonies at any dose level with or without the metabolic activation using either incorporation method.

All of the positive controls induced marked increases in the frequency of the revertant colonies confirming the sensitivity of the bacterial strains and the activity of the S9 mix.

Under the conditions of the test, the test item ‘reaction mass of mixed xylenes and sulphur monochloride’ was considered to be non-mutagenic.