1-(2-methyl-4-(2-methylphenylazo)phenylazo)-2-naphthol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Similar to OECD 471

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity Testing of Certified Food Colors and Related Azo, Xanthene And Triphenylmethane Dyes with the Salmonella/Microsome System

Author:

Joseph P. Brown

Year:

1978

Bibliographic source:

Mutation Research

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Similar to OECD 471

Principles of method if other than guideline:

Ames assay was performed to determine the mutagenic nature of Test chemical .

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(2-methyl-4-(2-methylphenylazo)phenylazo)-2-naphthol

EC Number:

201-635-8

EC Name:

1-(2-methyl-4-(2-methylphenylazo)phenylazo)-2-naphthol

Cas Number:

85-83-6

Molecular formula:

C24H20N4O

IUPAC Name:

1-(2-methyl-4-(2-methylphenylazo)phenylazo)-2-naphthol

Details on test material:

- Name of test material: Sudan IV
- Molecular formula: C24H20N4O
- Molecular weight: 380.449 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data
- Smiles: c12c(\N=N\c3c(cc(\N=N\c4c(cccc4)C)cc3)C)c(ccc1cccc2)O

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA100, TA1537, TA1538, TA98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver homogenate fraction "S-9" was prepared from the liver of female Sprague Dawley rats injected i.p. with about 0.5 ml of a corn oil solution of Aroclor 1254 (400 mg/ml) to give a dose of 500 mg/kg body weight.

Test concentrations with justification for top dose:

100 or 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test materail was soluble in DMSO

Controls

Untreated negative controls:

yes

Remarks:

Sodium dithionite

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

ethylmethanesulphonate

methylmethanesulfonate

other: Anthragallol and 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 3 days
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The criteria adopted for scoring a mutagenic response in routine plate tests was the observed number of revertants should exceed twice the background value for that given assay and exceed the 99.9% confidence limit based on historical controls.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537, TA1538 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Without reduction performed using Sodium dithionite

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

For TA1538 at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537, TA1538 and TA98

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

With reduction by sodium dithionite at 100 µg/plate

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium, other: TA1535 and TA100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

With reduction performed using sodium dithionite at 100 µg/plate

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium, other: TA1537, TA1538 and TA98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

With reduction performed using Sodium dithionite at 100 µg/plate

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The chemical reduction of Sudan IV was performed by dissolving the chemical in DMSO with an equal volume of freshly prepared aqueous dithionite solution of the same weight concentration.

Remarks on result:

other: No mutagenic effect were observed

Any other information on results incl. tables

Table: Screening of Sudan IV

Test concentration	Chemical reduction (dithionite)	Microsome activation	Number of His^+revertants/plate
			TA1535	TA100	TA1537	TA1538	TA98
500µg/plate	-	-	46	80	8	12*	22
		+	35	115	14	48	50
100µg/plate	+	-	24	110	10	23	19
		+	17	105	35	303	400

*: Microbial toxicity

Applicant's summary and conclusion

Conclusions:

Test chemical failed to induce mutation in Salmonella typhimurium strain TA1535, TA100, TA1537, TA1538 and TA98 in the presence and absence of S9 metabolic activation system without reduction using sodium dithionite at 500 µg/plate and without S9 activation in the same strains with reduction being performed at 100 µg/plate and also in strains TA1535 and TA100 in the presence of S9 metabolic activation system with reduction at 100 µg/plate. It however induced mutation in the strains TA1537, TA1538 and TA98 in the presence of S9 metabolic activation system with reduction at 100 µg/plate. Based on the results of the study, Sudan IV does not induce mutation without reduction being performed using sodium dithionite in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Ames assay was performed to determine the mutagenic nature of test chemical in the presence and absence of S9 metabolic activation system. The study was also performed to determine the mutagenic nature of component amines formed upon chemical reduction. Non water soluble Sudan IV was dissolved in DMSO ans mixed with an equal volume of freshly prepared aqueous dithionite solution of the same weight concentration for reduction. The plates were incubated at 37 °C for 3 days prior to determining the number of revertants/plate. The criteria adopted for scoring a mutagenic response in routine plate tests was that the observed number of revertants should exceed twice the background value for that given assay and exceed the 99.9% confidence limit based on historical controls. Test chemical failed to induce mutation in Salmonella typhimurium strain TA1535, TA100, TA1537, TA1538 and TA98 in the presence and absence of S9 metabolic activation system without reduction using sodium dithionite at 500 µg/plate and without S9 activation in the same strains with reduction being performed at 100 µg/plate and also in strains TA1535 and TA100 in the presence of S9 metabolic activation system with reduction at 100 µg/plate. It however induced mutation in the strains TA1537, TA1538 and TA98 in the presence of S9 metabolic activation system with reduction at 100 µg/plate. Based on the results of the study, test chemical does not induce mutation without reduction being performed using sodium dithionite in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.