2,6-Diamino-4,5,6,7-tetrahydrobenzothiazole

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 2 august to 9 september 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in GLP compliance and in accordance with several internationally established guidelines (OECD, EEC, EPA guidelines, see below).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6-Diamino-4,5,6,7-tetrahydrobenzothiazole

Cas Number:

104617-49-4

Molecular formula:

C7H11N3S

IUPAC Name:

2,6-Diamino-4,5,6,7-tetrahydrobenzothiazole

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): SND 855 BS; 2,6-Diamino-4,5,6,7-tetrahydrobenzothiazole
- Physical state: beige crystalline powder
- Analytical purity: 97.9 % (HPLC)
- Purity test date: 12 July 1994
- Lot/batch No.: Krö 27
- Expiration date of the lot/batch: Sept. 1994
- Storage condition of test material: At ambient temperature in the dark

Method

Target gene:

TA1535; hisG46, uvrB, rfa
TA 1537; hisC3076, uvrB, rfa
TA 100; hisG46, uvrB, rfa, R fact.
TA 98, hisD3052, uvrB, rfa, R fact.

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

nitroreductase deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat liver)

Test concentrations with justification for top dose:

10, 100, 500, 1500, 3000, 5000 µg/plate of the test substance

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: 1-methyl-3-nitro-1-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation

DURATION
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: 3 each per concentration level and control

Evaluation criteria:

At the present time, the most common method of evaluation of data from this short-term mutagenicity test is the arbitrary rule that a doubling or, in the case of strains with low background counts, a tripling of the spontaneous reversion rate at one or two test substance concentrations constitues a positive response.

Statistics:

not regarded as neccessary according to the OECD guidelines.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

other: TA 1537, TA 98 and TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Induction of mutations in the absence of a metabolizing system

Substance	Conc. (µg/pl.)	Number of revertants per plate
		TA 1535	TA 1537	TA 100	TA 98
Solvent control (methanol, 100 µl/pl.) (Experiment 1: mean value; standard deviation)		6 (2)	7 (1)	95 (11)	14 (4)
(Experiment 2: mean value; standard deviation)		12 (3)	7(2)	96 (6)	15 (4)
SND 919 intermediate 2 (Experiment 1: mean value; standard deviation)	10	7 (3)	6 (3)	110 (12)	15 (1)
	100	8 (1)	8 (4)	105 (1)	18 (2)
	500	7 (1)	7(1)	115 (8)	24 (2)
	1500	12 (5)	9 (4)	105 (1)	22 (5)
	3000	8 (6)	11 (3)	120 (32)	26 (3)
	5000	6 (3)	0 (0)	127 (9)	27 (6)
MNNG	2	>2000	1243	1148	1124
9AA	80	>2000	1453	1291	1617
2NF	5	>2000	688	1267	1258
(Experiment 2: mean value; standard deviation)	10	9 (4)	9 (3)	101 (19)	16 (1)
	100	7 (5)	10 (2)	103 (14)	19 (4)
	500	8 (1)	7 (2)	103 (23)	18 (7)
	1500	8 (5)	10 (1)	99 (8)	19 (6)
	3000	10 (1)	10 (6)	118 (9)	23 (4)
	5000	11 (4)	0 (0)	112 (24)	30 (4)
MNNG	2	>2000	772	927	1234
9AA	80	>2000	964	954	1341
2NF	5	>2000	523	823	1343

Induction of mutations in the presence of a metabolizing system

Substance	Conc. (µg/pl.)	Number of revertants per plate
		TA1535	TA1537	TA100	TA98
Solvent control (methanol 100 µl/pl.) (Experiment 1: mean value; standard deviation)		10 (2)	10 (3)	106 (3)	27 (3)
(Experiment 2: mean value; standard deviation)		9 (3)	7 (2)	109 (11)	28 (0)
SND 919 intermediate 2	10	9 (2)	9 (4)	97 (6)	28 (6)
	100	8 (3)	8 (4)	129 (14)	56 (3)
	500	11 (3)	19 (6)	138 (24)	173 (36)
	1500	10 (2)	33 (2)	171 (18)	480 (40)
	5000	11 (6)	27 (1)	187 (19)	646 (28)
2AA	0.5; 2.5 (2*)	103 (28)	130 (19)	257 (18)	188 (10)

The substance under investigation was tested at a concentration range of 10 to 5000 µg/plate. The experiments were done with and without metabolic activation with rat liver homogenates. The experiments were repeated once.

The mutation frequencies observed on the plates without substance were all within the spontaneous range known for the tester strains.

The findinds with the positive control substances confirmed the known sensitivity of the tester strain and, in the case of 2 -aminoanthracene, the full activity of the metabolizing system.

In the absence of S9 mix, no mutagenicity of the test substance was observed in the strains TA1535, TA1537 and TA100, whereas a marginal increase over the negative control value of just twice the spontaneous mutation frequency was seen in strain TA98 at a concentration of 5000 µg/plate. In the presence of S9 mix, the test substance was not mutagenic in strain TA1535, but increases in mutation frequencies were seen in a concentration range of 100 to 5000 µg/plate in the remaining strains, TA 1537, TA 100 an TA 98. Of these three tester strains, the highest induced mutation frequencies were found in strain TA98: the dose related increase in revertants ranged from 2 times (at 100 µg/plate) to about 24 times (at 5000 µg/plate) the spontaneous frequency.

The test substance was bacteriotoxic in strain TA1537 at concentrations >3000 µg/plate in the absence of S9 mix.

Applicant's summary and conclusion

Conclusions:

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

It was concluded that the sample of 2,6-diamino-4,5,6,7-tetrahydrobenzothiazole tested was mutagenic in the standard plate incorporation assay. The (marginal) effect seen in the absence of a metabolizing system was greatly enhanced by the addition of rat liver homogenates.