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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES Assay;

Test chemical  failed to induce mutation in Salmonella typhimurium strain TA100, TA98, TA1535, TA1537 and TA1538 with and without S9 metabolic activation system in the spot test performed and hence is not likely to classify for gene mutation in vitro.

Chromosome aberration study in mammalian cell

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical1 and test chemical 2. The study was performed using CHO-WBL cells in the presence and absence of exogeneous metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels upto 5 mg/mL. In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay in most instances when cell cycle delay was observed in the SCE assay. In these tests the cell growth period was extended to about 20 hr. Cells were harvested. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed and the chromosomal aberrations were scored. The test chemical did not induce chromosomal aberrations when tested to toxicity. Precipitate was evident at doses of 250µg/ml and above for the test chemical 1. Based on the observations made, the test chemical 1 and test chemical 2 did not induce chromosome aberrations in the CHO-WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

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:

comparable to guideline study

Remarks:

Similar to OECD 471

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation by the spot assay was performed for Test chemical.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

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:

S9 metabolic activation system isolated female Sprague Dawley rats given ip injection of Aroclor 1254 dissolved in corn oil

Test concentrations with justification for top dose:

200 µg/Plate

Vehicle / solvent:

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

Untreated negative controls:

not specified

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, 2-anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: Spot test

DURATION
- Preincubation period: No data
- Exposure duration: 72 hrs
- Expression time (cells in growth medium): 72 hrs
- 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

NUMBER OF CELLS EVALUATED: No data

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

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: 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 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 our historical controls.

Statistics:

No data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic effect were observed

Table: Screening of Sudan IV

Test concentration	Chemical reduction (dithionite)	Microsome activation	Number of His^+revertants/plate
			TA1535	TA100	TA1537	TA1538	TA98
200µg/plate	-	-	-	-	-	-	-
		+	-	-	-	-	-

Conclusions:

Test chemical failed to induce mutation in Salmonella typhimurium strain TA100, TA98, TA1535, TA1537 and TA1538 with and without S9 metabolic activation system in the spot test performed and hence is not likely to classify for gene mutation in vitro.

Executive summary:

Gene mutation by the spot assay was performed for test chemical was used at dose level of 200 µg/plate both with and without S9 metabolic activation system. The plates were incubated for 3 days at 37°C for the growth of colonies to occur. The plates were observed for an increase in the number of His+ revertants. 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 our historical controls. Concurrent positive control was also included in the study test chemical failed to induce mutation in Salmonella typhimurium strain TA100, TA98, TA1535, TA1537 and TA1538 with and without S9 metabolic activation system in the spot test performed and hence is not likely to classify for gene mutation in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL 1/2

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 fractions (livers of Aroclor 1254-treated male Sprague-Dawley rats.)

Test concentrations with justification for top dose:

1. -S9 (Harvest time: 10 hrs): 0, 2500, 3850, 5000 µg/mL
+S9 (Harvest time: 12 hrs): 0, 2500, 3850, 5000 µg/mL

2. Without activation: 15.1000, 37.8000, 75.5000 µg/ml
With activation: 125.000, 165.000, 250.000 µg/ml

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical is soluble in DMSO

2. - Vehicle(s)/solvent(s) used: Medium
- Justification for choice of solvent/vehicle: The test chemical is soluble in medium

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Medium

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

1/2.
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration:
- S9: 8 hrs
+ S9: 2 hrs
- Expression time (cells in growth medium): 8 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): -S9: 10 hrs, +S9: 12 hrs

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

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: One hundred to 200 cells from each of the three highest scorable doses were analyzed

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: No data available

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

1./2. All aberrations were individually classified (e.g., chromatid breaks, chromosome breaks, triradials, etc.). These data were combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations. Only the total percent cells with aberrations was considered in the statistical evaluation. Gaps and endoreduplications were recorded but were not included in the statistical analyses.

Statistics:

1./2. Trend test.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL 1./2.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

1./2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.0 – 7.5
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Chemicals were tested up to 5 mg/ml or as limited by solubility and/or toxicity. Solubility tests were conducted to determine dose range and choice of solvent (water, dimethyl sulfoxide, acetone, or ethanol, in that order of preference). In the assays for chromosomal aberrations, the top dose (TD) was based on toxicity, solubility, or the upper testing limit (5 mg/ml). The doses used were generally the TD, 0.75 TD, 0.50 TD, 0.25 TD, 0.1 TD, 0.075 TD, 0.05 TD, and 0.025 TD. The highest three doses with a sufficient number of cells were analyzed for chromosomal aberrations

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosome aberrations in the CHO WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical1 and test chemical 2. The study was performed using CHO-WBL cells in the presence and absence of exogeneous metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels upto 5 mg/mL. In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay in most instances when cell cycle delay was observed in the SCE assay. In these tests the cell growth period was extended to about 20 hr. Cells were harvested. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed and the chromosomal aberrations were scored. The test chemical did not induce chromosomal aberrations when tested to toxicity. Precipitate was evident at doses of 250µg/ml and above for the test chemical 1. Based on the observations made, the test chemical 1 and test chemical 2 did not induce chromosome aberrations in the CHO-WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce chromosome aberrations in the CHO WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data for the various test chemicals was reviewed to determine the mutagenic nature of 1-(2-methyl-4-(2-methylphenylazo)phenylazo)-2-naphthol (85-83-6). The studies are as mentioned below:

 

AMES Assay;

Gene mutation by the spot assay was performed for test chemical was used at dose level of 200 µg/plate both with and without S9 metabolic activation system. The plates were incubated for 3 days at 37°C for the growth of colonies to occur. The plates were observed for an increase in the number of His+ revertants. 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 our historical controls. Concurrent positive control was also included in the study test chemical failed to induce mutation in Salmonella typhimurium strain TA100, TA98, TA1535, TA1537 and TA1538 with and without S9 metabolic activation system in the spot test performed and hence is not likely to classify for gene mutation in vitro.

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.

Gene mutation by the preicubation assay was performed for Sudan IV. Sudan IV was used at dose levels of 0, 5, 50, 500, 1000 or 5000 µg/plate using Salmonella typhimurium strain TA100, TA98 with and without S9 metabolic activation system. The plates were incubated for 2 days at 37°C and observed for a dose dependent increase in the number of His+ revertants/plate. Pure Sudan IV failed to induce mutation in Salmonella typhimurium strain TA100, TA98 with and without S9 metabolic activation system in the preincubation assay performed and hence is not likely to classify for gene mutation in vitro.

Chromosome aberration study in mammalian cell

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using CHO-WBL cells in the presence and absence of exogeneous metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels upto 5 mg/mL. In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay in most instances when cell cycle delay was observed in the SCE assay. In these tests the cell growth period was extended to about 20 hr. Cells were harvested. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed and the chromosomal aberrations were scored. The test chemical did not induce chromosomal aberrations when tested to toxicity. Precipitate was evident at doses of 250µg/ml and above. Based on the observations made, the test chemical did not induce chromosome aberrations in the CHO-WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using CHO-WBL cells in the presence and absence of exogeneous metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels upto 5 mg/mL. In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay in most instances when cell cycle delay was observed in the SCE assay. In these tests the cell growth period was extended to about 20 hr. Cells were harvested. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed and the chromosomal aberrations were scored. The test chemical did not induce chromosomal aberrations when tested to toxicity. Based on the observations made, the test chemical did not induce chromosome aberrations in the CHO-WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data summarized, 1-(2-methyl-4-(2-methylphenylazo)phenylazo)-2-naphthol (85-83-6 )did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Based on the above annotation and CLP criteria ,test chemical ,1-(2-methyl-4-(2-methylphenylazo)phenylazo)-2-naphthol (85-83-6 )did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.