1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole

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:

May - June 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The only difference to guideline was that the repeat experiment was not performed by pre-incubation method but rather as a second plate-incorporation test. Study was performed under GLP-like quality control.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

no

Remarks:

The only difference to guideline was that the repeat experiment was not performed by pre-incubation method but rather as a second plate-incorporation test. Study was performed under GLP-like quality control.

GLP compliance:

no

Remarks:

but performed under GLP like quality assurance with QAU statement included.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole

EC Number:

401-280-0

EC Name:

1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole

Cas Number:

91273-04-0

Molecular formula:

C19 H38 N4

IUPAC Name:

bis(2-ethylhexyl)[(1H-1,2,4-triazol-1-yl)methyl]amine

Details on test material:

- Analytical purity: commercial grade

Method

Target gene:

his

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

0.08 - 5000 µg/plate in the toxicity test.
20, 78, 313, 1250 and 5000 µg/plate in the mutagenicity test.

Vehicle / solvent:

Acetone

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA 98: daunorubicin-HCl; TA 100: 4-nitroquinoline-N-oxide; TA 102: mitomycin-C; TA 1535: sodium azide; TA 1537: 9(5)-aminoacridine hydrochloride monohydrate

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA 98, TA 100, TA 1537, TA 102: 2-aminoanthracene; TA 1535: cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

- Exposure duration: 48 h

NUMBER OF REPLICATIONS: without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). In order to confirm the results the experiments were repeated.

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in the colony count

Evaluation criteria:

The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was carried out with the concentrations ranging from 0.08 to 5000 µg/0.1 ml. Thereafter, the concentration of 5000 µg/0.1 ml was used as the highest in the mutagenicity test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A growth-inhibiting effect of the test item occurred in the experiments without microsomal activation at the upper concentrations (cytotoxicity started to occur at 313 or 1250 µg/plate, depending on test strain). In the experiments with microsomal activation this effect was less pronounced (cytotoxicity started to occur at 1250 and/or 5000 µg/plate, depending on test strain).

Any other information on results incl. tables

Experiment I

	TA 98		TA 100		TA 102		TA 1535		TA 1537
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	22	37	185	204	230	263	16	18	10	15
20	25	36	176	191	186	264	15	19	4	11
78	28	41	166	251	178	257	14	18	7	11
313	23	44	171	192	71	225	8	15	6	16
1250	16	26	95	145	17	90	0	13	0	0
5000	0	0	0	0	0	0	0	4	0	0
solvent control	20	45	171	170	210	275	13	19	9	10
positive control A	264	1148	665	608	887	907	470	353	684	98
positive control B	668		1215		1255		387		1597

Experiment II

	TA 98		TA 100		TA 102		TA 1535		TA 1537
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	24	47	158	171	322	298	13	17	8	20
20	31	47	143	153	298	344	12	13	11	15
78	28	40	183	179	276	326	14	12	8	11
313	20	34	158	165	215	263	9	12	10	14
1250	5	29	92	172	0	52	0	3	0	1
5000	0	0	0	0	0	0	0	0	0	0
solvent control	20	45	160	157	256	332	12	16	7	15
positive control A	442	1451	670	1489	797	2170	246	300	558	262
positive control B	649		1286		992		528		1520

Positive Controls

Without S9 mix:

TA 98: daunorubicin-HCl, A: 5 and B: 10 µg/0.1 mL phosphate buffer

TA 100: 4-nitroquinoline-N-oxide, A: 0.125 and B: 0.25 µg/0.1 mL phosphate buffer

TA 102: mitomycin-C, A: 0.5 and B: 1.0 µg/0.1 mL bidistilled water

TA 1535: sodium azide, A: 2.5 and B: 5.0 µg/0.1 mL bidistilled water

TA 1537: 9(5)- aminoacridine hydrochloride monohydrate, A: 50 and B: 100 µg/0.1 mL DMSO

With S9-Mix:

TA 98, TA 100, TA 1537: 2-aminoanthracene, 5 µg/0.1 mL DMSO

TA 102: 2-aminoanthracene, 20 µg/0.1 mL DMSO

TA 1535: cyclophosphamide, 250 µg/0.1 mL phosphate buffer

Applicant's summary and conclusion

Conclusions:

Interpretation of results: Negative with and without metabolic activation

Based on the presented results and under the conditions employed, the test article did not induce point mutations in presence or absence of a metabolic activation system and is therefore regarded as not mutagenic in the Ames test.

Executive summary:

In order to investigate the test article's potential to cause point mutation in bacteria, an AMES test similar in design to the OECD guideline No. 471 was carried out with the tester strains Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test article was applied by the plate incorporation method at concentrations of 20, 78, 313, 1250 and 5000 µg/0.1 ml either with or without a metabolic activation system (rat liver S9 mix). The experiment was performed in triplicates and repeated once for confirmation. Positive controls were performed in parallel to check the tester strains sensitivity. In none of the experiments did treatment with the test substance lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the controls. A growth-inhibiting effect of the test item occurred in the experiments without microsomal activation at the upper concentrations (cytotoxicity started to occur at 313 or 1250 µg/plate, depending on test strain). In the experiments with microsomal activation this effect was less pronounced (cytotoxicity started to occur at 1250 and/or 5000 µg/plate, depending on test strain). In conclusion, no evidence of the induction of point mutations by the test substance or by its metabolites formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the substance is considered as not mutagenic in this assay.