Reaction mass of N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo]naphthalen-2-amine and N-(2-ethylhexyl)-1-[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]naphthalen-1-amine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Gene mutation toxicity study of the test chemical

Author:

Zeiger et al

Year:

1987

Bibliographic source:

Environmental Mutagenesis

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Bacterial reverse mutation assay was performed to evaluate the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(1,4-dimethylpentyl)-p-phenylenediamine

EC Number:

221-375-9

EC Name:

N,N'-bis(1,4-dimethylpentyl)-p-phenylenediamine

Cas Number:

3081-14-9

Molecular formula:

C20H36N2

IUPAC Name:

1-N,4-N-bis(5-methylhexan-2-yl)benzene-1,4-diamine

Details on test material:

- Name of test material: N,N'-Bis-(1,4 dimethylpentyl)-p-phenylenediamine
- IUPAC name: 1-N,4-N-bis(5-methylhexan-2-yl)benzene-1,4-diamine
- Molecular formula: C20H36N2
- Molecular weight: 304.518 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, and TA97

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 S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared

Test concentrations with justification for top dose:

0, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 33, 100 or 333 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble and stable in DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene (2-AA) (All strains; +S9), 4-nitro-o-phenylenediamine (TA98; -S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic C+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose-related increase was judged insufficiently high to justify a call of "+W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen.

The chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials.

It chemical was considered to be questionable (?) if a reproducible increase of his+ revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA1535, TA100, TA97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: The chemical was tested initially in a toxicity assay to determine the appropriate dose range. The toxicity assay was performed by using TA100 or the system developed by Waleh et al. Toxic concentrations were those at which a decrease in the number of his+ colonies was seen or at which there was a clearing in the density of the background lawn

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

 Table: Mutagenicity information

Dose (µg/plate)	TA100
	-S9		10% HLI		30% HLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	105	4.7	104	2.9	112	2.0
0.03	107	5.3
0.1	104	12.8
0.3	109	4.2
1.0	100	0.6
3.0	92	8.1	130	2.8	122	5.3
10			105	4.0	117	5.3
33			117	5.2	139	1.9
100			105	6.2	132	1.9
333			132	9.0	130	7.8
Positive control	372	9.7	1521	82.5	645	9.0

 

Dose (µg/plate)	TA1535
	-S9		10% HLI		30% HLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	27	3.2	10	1.7	10	1.2
0.03
0.1
0.3	20	3.2
1.0	23	4.2
3.0	19	5.3	10	1.0	6	0.7
10	20	2.9	7	0.3	7	1.2
33	17	3.0	7	0.9	8	0.9
100			7	0.9	6	0.3
333			6	1.5	5	1.0
Positive control	372	9.7	1521	82.5	645	9.0

 

Dose (µg/plate)	TA1537
	-S9		10% HLI		30% HLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	4	1.2	5	0.3	6	0.6
0.03	4	0.6
0.1	5	1.8
0.3	5	1.8
1.0	3	1.3
3.0	7	0.6	8	0.3	5	0.7
10			6	1.7	6	0.7
33			6	1.2	5	1.5
100			5	0.9	8	0.3
333			7	1.5	4	1.0
Positive control	192	6.0	342	12.0	108	3.5

 

Dose (µg/plate)	TA98
	-S9		10% HLI		30% HLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	19	1.0	17	0.9	25	2.3
0.03
0.1
0.3	20	2.3
1.0	14	1.2
3.0	15	1.8	23	4.4	30	3.2
10	19	3.7	25	3.3	28	5.2
33	16	0.7	24	0.3	30	0.3
100			23	3.3	33	3.2
333			21	3.2	27	0.9
Positive control	844	36.7	1655	37.1	541	10.7

 

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce a reproducible, dose-related increase in his+ revertants over the corresponding solvent in the S. typhimurium tester strains TA100, TA97, TA1535 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs.

 

The test compound was dissolved in DMSO and was used at a dosage level of 0, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 33, 100 or 333 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study.

 

The test chemical did not induce a reproducible, dose-related increase in his⁺revertants over the corresponding solventin the S. typhimurium tester strains TA100, TA97, TA1535 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.