Dimethyl[4-[4-(dimethylamino)-α-phenylbenzylidene]-2,5-cyclohexadien-1-ylidene]ammonium hydrogen sulphate

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:

July to September 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Reference 4

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: ETAD toxicological method No. 005

Version / remarks:

1980

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

1. Ames, B.N.; Durston, W.W.; Yamasaki, E.; Lee, F.D.: Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Nat. Acad. Sci. USA, /2, 2281 - 2285 (1973)
2. Ames, B.N.; McCann, J.; Yamasaki, E.: Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mut. Res., 21, 347 - 364 (1975)
3. McCann, J.; Ames, B.N.: Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA, 72, 5135 - 5139 (1975)
4. McCann, J.; Ames, B.N.: Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals: Discussion. Proc. Nat. Acad. Sci, USA,
22, 950 - 954 (1976)
5. Purchase, I.F.H.; Longstaff, E.; Ashby, 3.; Styles, J.A.; Anderson, D.; Lefevre, P.A.; Westwood, F.R.: An evaluation of six short term tests for detecting organic chemical carcinogens Br. J. Cancer, 27, 837 - 959 (1978)
6. Ames, B.N.; Lee, F.D.; Durston, W.E.: An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Nat. Sci. USA, 70, 782 - 786 (1973)
7. McCann, J.; Spingarn, N.E.; Kobori, J.; Ames, B.N.: Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids. Proc. Nat. Acad. Sci. USA, E, 979 - 983 (1975)
8. Alvares, A.P.; Bickers, D.R.; Kappas, A.: Polychlorinated biphenyls: A new type of inducer of cytochrome P-448 in the liver. Proc. Nat. Acad. Sci. USA, 70, 1321 - 1325 (1973)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-methyl-, ethanedioate

EC Number:

241-922-5

EC Name:

Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-methyl-, ethanedioate

Cas Number:

18015-76-4

Molecular formula:

C25H26N2O4

IUPAC Name:

Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-methyl-, ethanedioate

Test material form:

solid: particulate/powder

Details on test material:

Basic Green 4

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat S9-mix induced with Aroclor 1254

Test concentrations with justification for top dose:

5, 10, 20, 50, 100, 500, 2500, 5000 µg/plate with S9
2.5, 5, 10 , 20, 50, 100, 500, 2500, 5000 µg/plate without S9

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

sterility check

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), 4-nitro-o-phenylendiamine

Details on test system and experimental conditions:

Standard Plate Test
Incubation at 37°C for 48 hours
Quadruple testing

Evaluation criteria:

Cytotoxicity: reduced growth rate or thinning of bacterial lawn
Positive: - dose-related and reproducible increase in number of revertant colonies
- doubling of spontaneous mutation rate in at least one tester strains either with or without S9

Statistics:

NA

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, 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:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Basic Green 4 is not mutagenic in this bacterial test system either with or without exogenous metabolic activation at the dose levels investigated.

Executive summary:

Compound T 2015 - 6 (Basic Green 4 oxalate) was tested for mutagenicity with the strains TA100, TA1535, TA1537, TA98 of Salmonella typhimurium according to the procedure developed by Ames and co-workers and following the instructions of the ETAD toxicological method No. 005 July 1980.

The studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. In a first step, a range of 20, 50, 100, 500, 2500, 5000 µg/plate was used. Due to the high cytotoxicity of Basic Green 4, additional dose levels of 5, 10, 20, and 50 µg/plate with S9 -mix and 2.5, 5, 10 , and 20 µg/plate without S9 -mix were tested.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: T 2015 -6 proved to be toxic to the bacteria and additional lower dose levels were added to the test. The test substance proved to be toxic at 2.5 µg/plate and above without S9 and at 20 µg/plate and above with S9. No bacterial growth was observed from 100 µg/plate onward with or without S9 -mix.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show an influence on the number of revertants in any of the bacterial strains. Also in the presence of metabolic activation system, treatment of the cells with T 2015 - 6 did not result in an increase in the number of the revertant colonies with any of the strains used.

Summarizing, it can be stated that T 2015 - 6 is not mutagenic in this bacterial test system either with or without exogenous metabolic activation at the dose levels investigated.