1,2-Benzisothiazole, 3-methyl-, 1,1-dioxide

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:

2016-08-15 to 2016-08-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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:

3-methyl-1λ⁶,2-benzothiazole-1,1-dione

EC Number:

812-907-6

Cas Number:

34989-82-7

Molecular formula:

C8H7NO2S

IUPAC Name:

3-methyl-1λ⁶,2-benzothiazole-1,1-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Lot: 216-106
- Expiry date: 2018-06-16
- Storage conditions: Keep at room temperature

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations.
The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Phenobarbital and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

5000, 1600, 500, 160, 50 and 16 μg/plate
Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: good solubility and guideline conform

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2AA), with metabolic activation in all strains; 4-nitro-1,2-phenylene-diamine (NPD), without metabolic activation in TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: colony and background lawn development

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control.
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined, the mean values, standard deviations and the mutation rates were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the Initial Mutation Test no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix); however in the Confirmatory Mutation Test (Pre-Incubation Test) heavy precipitate was noticed on the plates at the highest examined concentration of 5000 μg/plate in absence and presence of exogenous metabolic activation (±S9 Mix). The obtained precipitate disturbed the scoring of colonies and background lawn development; therefore the precipitated plates were evaluated additionally by microscope (at 40X magnification).

HISTORICAL CONTROL DATA
All observed colony number remained within the historical control values, except for TA100 without S9 at 5000 μg/plate in the Confirmatory Mutation Test. There a lower revertant colony number was observed, that remained without any biological significance.

Any other information on results incl. tables

Initial Mutation Test (Plate Incorporation)

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results without S9
Untreated	8.7 ± 5.13	6.7 ± 2.08	18 ± 1	104 ± 9.17	20 ± 0
DMSO	11.3 ± 4.04	5.3 ± 0.58	19 ± 1.73	85.7 ± 6.03	17 ± 3
Ultrapure Water Control	7.3 ± 1.15			102.7 ± 2.31	17.7 ± 3.79
5000	12.3 ± 1.53	4 ± 2.65	10 ± 0	78.7 ± 8.33	14.7 ± 2.31
1600	15.3 ± 3.21	6.7 ± 4.04	13.3 ± 3.51	88.7 ± 7.02	16.7 ± 3.06
500	15.7 ± 3.21	4.7 ± 3.79	17 ± 3.61	100 ± 17.32	19.3 ± 3.79
160	11.3 ± 4.16	4.7 ± 2.52	20.3 ± 4.73	97.3 ± 11.37	22 ± 3.61
50	13.3 ± 4.16	6 ± 5.29	13.7 ± 7.51	89.3 ± 12.22	23 ± 2
16	13 ± 1.73	3 ± 2.65	17.3 ± 4.62	101.3 ± 12.7	24 ± 8.54
NPD (4)			342.7 ± 26.1
SAZ (2)	672 ± 66.81			1245.3 ± 185.44
9AA (50)		750 ± 84.59
MMS (2 µL)					818.7 ± 118.68
	Results with S9
Untreated	12.3 ± 4.93	6.3 ± 1.63	21.7 ± 6.11	132 ± 11.14	29.3 ± 4.16
DMSO	14 ± 2.65	7.3 ± 3.06	21.7 ± 7.02	128 ± 18.33	28 ± 2
5000	11.3 ± 4.51	8.3 ± 5.03	20.3 ± 4.04	119.3 ± 11.72	21.7 ± 4.73
1600	12 ± 1	9.7 ± 3.21	24.7 ± 3.79	109.3 ± 11.02	21 ± 4.58
500	13.3 ± 5.86	8 ± 3.46	23 ± 2	117.3 ± 6.11	27.7 ± 9.07
160	14.7 ± 1.53	9 ± 5	22.3 ± 2.52	113.7 ± 14.84	29.7 ± 8.96
50	14.3 ± 2.89	10 ± 3.61	25 ± 2	120.7 ± 6.43	32 ± 6.56
16	10.3 ± 3.06	8.7 ± 3.51	24.3 ± 4.73	122.7 ± 9.87	33 ± 3
2AA (2)	199.3 ± 37.65	110 ± 17.06	1512 ± 246.45	2200 ± 381.58
2AA (50)					234 ± 15.62

NPD: 4-nitro-1,2-phenylene-diamine; SAZ: Sodium acide; 9AA: 9-aminoacridine; MMS: methylmethanesulfonate; 2AA: 2-aminoanthracene

 

 

Confirmatory Mutation Test (Pre-Incubation)

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results without S9
Untreated	9.3 ± 2.52	8 ± 2.65	17.7 ± 9.07	90.3 ± 13.2	19.3 ± 2.52
DMSO	11 ± 3	8 ± 4	16.7 ± 0.58	78 ± 9.64	24.3 ± 6.43
Ultrapure Water Control	11.7 ± 3.79			93.3 ± 6.66	28.3 ± 1.15
5000	15 ± 6.56	8.3 ± 4.73	11.7 ± 6.11	58 ± 7	17 ± 2.65
1600	11.3 ± 1.53	7.7 ± 4.73	16 ± 4.58	76.3 ± 8.74	21.3 ± 2.31
500	9.3 ± 1.53	9.7 ± 2.31	10 ± 0	73.7 ± 2.52	25.7 ± 8.08
160	14.3 ± 0.58	8.7 ± 4.04	12.3 ± 1.53	85.3 ± 11.59	25.3 ± 3.06
50	11.3 ± 3.79	8.3 ± 4.93	14 ± 2.65	74.3 ± 5.69	22.3 ± 115
16	10.7 ± 3.21	6.7 ± 4.62	15.7 ± 3.21	77 ± 2.65	16.7 ± 4.51
NPD (4)			254 ± 40.6
SAZ (2)	965.3 ± 53.27			1210.7 ± 122.46
9AA (50)		356 ± 107.63
MMS (2 µL)					962.7 ± 158.86
	Results with S9
Untreated	11.7 ± 0.58	7.7 ± 4.93	28.7 ± 3.51	129.7 ± 8.96	32.3 ± 4.51
DMSO	13.3 ± 3.21	6.3 ± 3.06	27.7 ± 2.89	108.7 ± 17.62	27.7 ± 4.04
5000	9.7 ± 4.51	7.3 ± 4.93	17.7 ± 1.53	79.3 ± 8.74	24 ± 5
1600	11 ± 3.61	7.3 ± 3.51	16.7 ± 2.52	88 ± 11.27	26.7 ± 4.93
500	11.7 ± 4.04	6 ± 3.46	18 ± 1.73	90.7 ± 12.66	27.3 ± 4.16
160	12.3 ± 3.21	8.3 ± 1.53	23.7 ± 1.53	90 ± 7	38.3 ± 4.62
50	9.7 ± 2.89	9 ± 3.61	22.3 ± 6.81	101.7 ± 7.51	37.3 ± 7.51
16	11.3 ± 3.51	6.3 ± 3.21	17 ± 7	98.7 ± 4.73	38 ± 4.36
2AA (2)	138 ± 7.81	164.7 ± 9.02	1117.3 ± 120.09	1114.7 ± 360.65
2AA (50)					170.3 ± 15.57

NPD: 4-nitro-1,2-phenylene-diamine; SAZ: Sodium acide; 9AA: 9-aminoacridine; MMS: methylmethanesulfonate; 2AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions the test substance was considered non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

The mutagenicity of the substance was studied with four mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in Escherichia coli WP2 uvrA according to OECD 471. The study was performed as two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations (5000, 1600, 500; 160; 50 and 16 μg/plate), including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test substance at any concentration level, either in the presence or absence of metabolic activation. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. In the Confirmatory Mutation Test (Pre-Incubation Test) heavy, interfering precipitate was noticed on the plates in the examined bacterial strains at the highest examined concentration level of 5000 μg/plate in absence and also in the presence of exogenous metabolic activation (±S9 Mix). The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test substance is considered non-mutagenic in this bacterial reverse mutation assay.