3,3'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[N-cyclohexyl-2,4,6-trimethylbenzenesulphonamide]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No indication of a significant mutagenic potential was detected in any of the tester strains used.

Link to relevant study records

Reference

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:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to guideline and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

Test concentrations with justification for top dose:

0; 33; 1 00; 333; 1 000; 2500; and 5000 ~glplate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA;

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: background lawn

Evaluation criteria:

A test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced. A test item producing neither a dose related increase in the number of revertants, nor a biologically relevant positive response at any one of the test points is considered nonmutagenic in this system.
A mutagenic response is described as follows:
A test item is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate (3,4).
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

not required

Species / strain:

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:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

without S9 mix

Concentration µg/plate	Revertants/plate (mean from three plates)
	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
	i	ii	i	ii	i	ii	i	ii	i	ii
Negative	14	13	6	8	23	26	115	128	40	40
Solvent	11	11	7	7	21	21	99	117	45	46
Positive*	1210	704	46	48	197	137	1202	801	1031	389
33	12	5	10	6	25	16	104	113	40	46
100	18	6	12	6	25	17	101	111	45	47
333	15	7	10	9	26	13	106	124	42	56
1000	15	9	8	12	29	18	111	115	40	47
2500	13	6	11	6	25	20	104	123	48	36
5000	13	5	9	7	28	17	106	98	55	34

 

With S9 mix

Concentration µg/plate	Revertants/plate (mean from three plates)
	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
	i	ii	i	ii	i	ii	i	ii	i	ii
Negative	16	13	16	7	34	23	124	154	44	42
Solvent	12	12	16	9	33	16	107	152	46	39
Positive**	196	81	85	68	624	429	973	746	523	219
33	14	8	18	8	32	15	108	160	42	36
100	13	10	15	6	35	19	116	173	49	39
333	14	11	19	7	34	17	129	157	44	46
1000	15	8	19	5	43	18	131	150	44	44
2500	16	8	17	8	47	23	131	139	54	43
5000	17	5	15	7	46	24	125	170	47	37

 

* Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100

4-nitro-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate)

Methyl methane sulfonate (5 µg/plate) strain WP2 uvrA

** 2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98, and TA 100

2-aminoanthracene (10.0µg/plate) strain WP2 uvrA

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce gene mutations in a bacterial reverse mutation assay (Ames) with and without metabolic activation.

Executive summary:

This study was performed to investigate the potential of the test material to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

33; 1 00; 333; 1 000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µglplate with and without metabolic activation in both independent experiments.

Minor toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in strain TA 1535 at 5000 µglplate in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore,the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

No indication of a significant mutagenic potential was detected in any of the tester strains used with or without metabolic activation in a bacterial reverse mutation assay (Ames Test).

Justification for selection of genetic toxicity endpoint
Study according to guideline and GLP; full report available

Justification for classification or non-classification

As not mutagenic potential was detected, classification is not warranted.