2,4,6-trinitrobenzene-1,3,5-triamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, K, rel. 2): non mutagenic up to cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 & E.coli WP2.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

purity of test substance, no. of cells per culture, no. of replications, vehicle, signs of toxicity, evaluation and interpretation of results, individual plate counts not reported

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

purity of test substance, no. of cells per culture, no. of replications, vehicle, signs of toxicity, evaluation and interpretation of results, individual plate counts not reported

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

pre-GLP

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan for S. typhimurium and E. Coli, respectively.

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-stimulated, rat liver-homogenate metabolic activation system was used

Test concentrations with justification for top dose:

5, 10, 50, 100, 500, 1000, 2500 and 5000 μg/plate, with and without S9-mix in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 & TA 100 and E. coli WP2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: 6-propiolactone (50 μg/plate for TA 1535); 2-Anthramine (20 μg/plate for TA 98, TA 100 and WP2)

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: S. typhimurium strains were obtained from Dr. Bruce Ames of the University of California at Berkeley. E. coli WP2 was obtained from Dr. D. McCalla.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Plates were incubated at 37 °C for 2 days.

Evaluation criteria:

None

Statistics:

None

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

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

Key result

Species / strain:

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

Table 7.6.1/1: Ames test - results

Test substance	Dose levels (µg/plate)	Mean no. Of revertants per plate
		Histidine revertants					Tryptophan revertants
		TA 1535	TA 1537	TA 1538	TA 98	TA 100	E. coli WP2
Without metabolic activation
Negative control	-	23	10	14	29	160	76
TATB	5	41	13	15	28	151	-
	10	30	7	12	32	126	81
	50	32	7	13	25	148	93
	100	35	8	16	21	148	78
	500	40	8	9	25	146	95
	1000	25	10	14	25	125	69
	2500	31	8	9	28	152	84
	5000	33	8	14	30	125	87
Positive control	*	680	>2000	310	40	160	72
With metabolic activation
Negative control	-	17	11	24	57	156	81
TATB	5	23	7	19	34	128	-
	10	20	9	28	25	151	91
	50	24	10	22	42	138	108
	100	22	7	20	39	132	87
	500	20	13	21	34	145	88
	1000	18	6	17	35	139	88
	2500	20	8	16	27	143	88
	5000	20	6	15	33	173	94
Positive control	**	-	-	-	>2000	>2000	1170

*Without metabolic activation - 9-Aminoacridine (100 μg/plate for TA 1537); 2-Nitrofluorene (50 μg/plate for TA 1538); 6-propiolactone (50 μg/plate for TA 1535); 2-Anthramine (20 μg/plate for TA 98, TA 100 and WP2)

**With metabolic activation - 2-Anthramine (20 μg/plate for TA 98, TA 100 and E. coli WP2)

Conclusions:

Under the test conditions, test substance is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA 1538, TA98 and TA100) and E. coli WP2 strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD Guideline 471, strains of Salmonella typhimurium (TA1535, TA1537, TA 1538, TA98 and TA100) and Escherichia coli WP2 were exposed to test substance at the following concentrations using the plate incorporation method: 

5, 10, 50, 100, 500, 1000, 2500 and 5000 μg/plate, with and without S9-mix

 

Negative and positive control groups were also included in mutagenicity tests.

 

The mean numbers of revertant colonies are fell within acceptable ranges for negative control treatments, and were elevated by positive control treatments.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. 

 

Under the test conditions, test substance is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA 1538, TA98 and TA100) and E. coli WP2 strains.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Newell, 1976	Ames Test (OECD 471) K, rel. 2	Gene mutation	TA 1535, TA 1537, TA 1538, TA 98, TA 100, E. coli WP2	-S9 +S9	Up to 5000 µg/plate	-S9 : non mutagenic +S9 : non mutagenic

 

Gene mutation Assay (Test n° 1):

A Bacterial Reverse mutation Assay (Ames test) was performed similarly to OECD guideline No. 471. Strains of S. typhimurium and E. Coli were exposed to the test substance at the following concentrations using the plate incorporation method: 5, 10, 50, 100, 500, 1000, 2500 and 5000 µg/plate with and without metabolic activation. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation.The mean numbers of revertant colonies are fell within acceptable ranges for negative control treatments, and were elevated by positive control treatments.The substance is therefore considered as non-mutagenic according to the Ames test.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.