4,5,6,7-tetrahydro-1H-benzotriazole

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:

03 Feb 2017 - 16 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,5,6,7-tetrahydro-1H-benzotriazole

EC Number:

229-858-6

EC Name:

4,5,6,7-tetrahydro-1H-benzotriazole

Cas Number:

6789-99-7

Molecular formula:

C6H9N3

IUPAC Name:

4,5,6,7-tetrahydro-1H-1,2,3-benzotriazole

Details on test material:

Physical state, appearance: solid, white
Storage conditions: room temperature

Method

Target gene:

his, trp

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:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 3000 and 6000 μg/plate
Due to the purity of the test substance 6.0 mg/plate was used as top dose in all experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Controls

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours in the dark

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn

POSITIVE CONTROLS

With S9 mix
• 2-aminoanthracene (2-AA)
- 2.5 μg/plate, dissolved in DMSO, strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO, strain: Escherichia coli WP2 uvrA

Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO, strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD)
- 10 μg/plate, dissolved in DMSO, strain: TA 98
• 9-aminoacridine (AAC)
- 100 μg/plate, dissolved in DMSO, strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO, strain: E. coli WP2 uvrA

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

HISTORICAL CONTROL DATA
The number of revertant colonies in the negative controls with and without S9 was within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the standard plate test only with S9 mix using the tester strains TA 1537 from about 3000 μg/plate onward and TA 98 at a concentration of 6000 μg/plate. In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants) was observed using TA 1537 without S9 mix at a concentration of 6000 μg/plate.

Any other information on results incl. tables

EXPERIMENTAL RESULT

Standard Plate Test

	Mean revertants per plate
Strain	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	10.7	10.3	96.0	106.7	7.7	12.0	21.3	31.3	23.3	21.0
33	10.3	8.3	108.3	111.7	10.3	12.0	16.7	25.7	27.0	26.0
100	11.7	10.3	113.7	118.7	7.3	9.7	17.7	34.7	20.3	23.7
333	13.7	12.7	100.3	118.7	10.3	9.3	16.0	19.3	22.7	29.3
1000	13.0	13.0	106.0	104.0	10.7	9.7	24.3	27.7	23.3	22.0
3000	14.0	14.0	115.3	107.3	7.7	7.0	21.7	21.3	22.7	28.3
6000	13.7	14.7	136.0	115.0	9.7	7.0	22.3	20.3	26.7	21.7
positive control	5382.7	188.7	4366.0	1330.3	1210.7	123.3	656.0	2513.0	1026.3	116.0

Preincubation Test

	Mean revertants per plate
Strain	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	7.7	10.3	80.3	94.7	6.7	8.7	20.0	32.3	23.0	23.7
33	10.7	5.0	86.0	86.0	6.3	11.0	18.3	36.7	30.3	18.7
100	8.7	9.3	94.3	85.0	7.0	8.7	19.0	23.7	23.7	17.3
333	9.7	9.3	86.0	89.0	7.3	11.3	20.0	31.3	16.7	24.0
1000	14.0	8.3	94.7	98.0	9.7	6.3	23.3	28.0	15.0	19.0
2600	14.3	13.0	85.7	103.3	6.7	11.0	15.7	27.0	20.7	24.3
5200	16.7	16.3	100.7	112.3	3.7	5.7	15.3	21.3	22.7	17.7
positive control	3495.3	228.3	2569.7	2330.7	1062.7	146.0	593.0	1850.0	606.3	103.0

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. The dose range tested was 33 μg - 6000 μg/plate both in the standard plate test and in the preincubation assay both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 3000 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.