(benzothiazol-2-ylthio)acetic acid

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:

1999-02-12 to 1999-03-16

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: in accordance with "Standards for Toxicity Investigations" .

Version / remarks:

Ministry of Labor, Notification No.77, September'l, 1988 and Notification No.67, June 2, 1997

GLP compliance:

yes

Remarks:

This study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September 1, 1988).

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(benzothiazol-2-ylthio)acetic acid

EC Number:

228-565-0

EC Name:

(benzothiazol-2-ylthio)acetic acid

Cas Number:

6295-57-4

Molecular formula:

C9H7NO2S2

IUPAC Name:

2-(1,3-benzothiazol-2-ylsulfanyl)acetic acid

Test material form:

other: white crystalline

Details on test material:

- Name of test material (as cited in study report): ABT or (2-Benzothiazolylthio)acetic acid
- Physical state: solid
- Appearance: white crystalline
- Analytical purity: 99%
- Purity test date: n.a.
- Lot/batch No.: 99001
- Expiration date of the lot/batch: n.a.
- Stability under test conditions: stable in ordinary temperature
- Storage condition of test material: stred at a cool and dark place

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:

rat liver S9 mix

Test concentrations with justification for top dose:

Dose finding test: with and without S9 mix: 4.88, 19.5, 78.1, 313, 1250, 5000 (µg/plate)
Main test with and without S9 mix: 313, 625, 1250, 5000 (µg/plate)
Confirmation test: with strain TA1537 with and without S9 mix: 313, 625, 1250, 5000 (µg/plate)

Positive controls without S9 mix:
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide: 0.01 µg/plate (TA100, WP2uvrA), 0.1 µg/plate (TA98)
NaN3: Sodium azide: 0.5 µg/plate (TA1535)
ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropy1amino]acridine' 2HCl: 1 µg/plate (TA 1537)

Positive controls with S9 mix:
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide: 0.01 µg/plate (TA100, WP2uvrA), 0.1 µg/plate (TA98)
NaN3: Sodium azide: 0.5 µg/plate (TA1535)
ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropy1amino]acridine' 2HCl: 1 µg/plate (TA 1537)
2AA: 2-Aminoanthracene: 0.5 µg/plate (TA98), 1 µg/plate (TA100), 2 µg/plate (TA1535, TA1537), 10 µg/plate (WP2uvrA)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Since the solubility of the test substance was less than 50 mg/mL in water but more than 50 mg/mL in DMSO, DMSO was selected as a solvent. It was confirmed that there was no change in the 50 mg/mL solution in DMSO, including color change and heat generation, until 2 hours after preparation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

The solvent used in the tests was employed as a negative control

Positive controls:

yes

Positive control substance:

sodium azide

other: 2(2-Furyl)3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

This study was performed by the pre-incubation method with and without S9 mix. Triplicate plates were used for the negative control group and duplicate plates per dose for the test substance treatment groups and the positive control groups. The test code number, name of test strain, presence or absence of S9 mix and dose level were noted on each plate.

Evaluation criteria:

The test substance was judged to be positive when the number of revertant colonies of the test substance treatment groups increased to twice or more that in the negative control with a concentration-dependent manner.

Statistics:

Any statistical methods were not used.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: TA 1535, TA 98, TA 100

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:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Result of Confirmation test: Number of revertant colonies in TA1537 strain was less than twice than in the negative control (with and without S9 mix). Increase of numbers of revertants in main test is juded accidential as value of negative group was low.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Confirmation test:
The number of revertant colonies in the test substance treatment groups was less than twice than in the negative control and in the groups with and without S9 mix in TA1537. Therefore, the increase of the number of revertant colonies in the main test was not confirmed dose-dependend and reproducible and due to the value of negative controls were low in the main test it was judged as an accidental increase result. The number of revertant colonies of the test substance treatment group in the main test was within the range of the historical data. From the above results, mutagenisity was judged to be negative in TA1537.

Remarks on result:

other: strain/cell type: salmonella typhimurium

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of mean number of revertants in Salmonella typhimurium strains and E. coli WP2uvrA strain with and without metabolic activation (mean of 3 plates for vehicle controls and 2 plates for test item treated or positive controls)

Results of dose finding Test
Concentration	TA100		TA1535		WP2uvrA		TA98		TA1537
[µg/plate]	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Negative Control	159	131	7	8	19	19	19	28	4	9
4.88	137	140	6	7	20	25	18	37	3	13
19.5	131	143	7	6	17	27	17	29	3	10
78.1	131	141	13	11	17	23	17	24	3	7
313	111	136	6	10	18	28	20	28	3	10
1250	117	113	10	5	19	22	21	25	4	13
5000	104	97	7	6	15	32	20	21	4	11
positive Control	AF-2	2AA	NaN3	2AA	AF-2	2AA	AF-2	2AA	ICR-191	2AA
	448	718	328	147	155	1050	585	291	2778	97
Results of Main Test
Concentration	TA100		TA1535		WP2uvrA		TA98		TA1537
[µg/plate]	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Negative Control	133	119	9	7	17	26	18	28	3	5
313	116	122	9	9	18	30	17	32	7	10
625	111	121	11	6	19	25	17	26	5	13
1250	106	114	7	7	21	23	19	24	8	17
2500	102	111	6	7	15	23	19	27	7	12
5000	92	91	5	7	19	23	20	20	6	11
positive Control	AF-2	2AA	NaN3	2AA	AF-2	2AA	AF-2	2AA	ICR-191	2AA
	449	766	386	163	149	1046	554	394	2599	99

AF-2:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide = positive control –S9 mix for strains TA100, WP2uvrA and TA98 [selected doses of 0.01, 0.01 and 0.1 µg/plate, respectively]

NaN₃: Sodium azide= positive control –S9 mix for strain TA1535 [selected dose:0.5 µg/plate]

ICR- 191:2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HCl = positive control –S9 mix for strain TA1537 [selected dose:1 µg/plate]

2AA: 2-Aminoanthracene = positive control + S9 mix for all tested bacteria strains[selected doses of 1, 2, 10, 0.5 and 2 µg/plate for strain TA100, TA1535, WP2uvrA, TA98 and TA1537, respectively]

 

Table 2: Confirmation Test: Summary of mean number of revertants in Salmonella typhimurium strain TA1537 with and without metabolic activation (mean of 3 plates for vehicle controls and 2 plates for test item treated or positive controls)

Results of confirmation Test
Concentration	TA1537
[µg/plate]	- S9	+ S9
Negative Control	8	10
313	10	10
625	9	11
1250	7	11
2500	10	11
5000	8	11
positive Control	ICR-191	2AA
	2753	110

ICR- 191:2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine'2HCl [Dose:1 µg/plate]

2AA: 2-Aminoanthracene[Dose: 2 µg/plate]

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the above results, it is concluded that the registered substance has no ability to induce mutation under the conditions of the present study.

Executive summary:

The ability of registered substance to induce mutations was investigated using Salmonella typhimurium strains TAl00, TA1535, TA98 and TAl537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix).

As a result, increase in the numbers of revertant colonies was not observed at any doses of the test substance treatment group in all test strains with and without S9 mix.

The numbers of revertant colonies in the negative and positive controls were confirmed to be within the range of the historical data in the testing facility. Based on the above results, it is concluded that the registered substance has no ability to induce mutation under the conditions of the present study.