N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

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:

May 97 - March 99

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial Reverse Mutation Test”, adopted 21st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EEC directive 92/69 at the time this study was done.

Deviations:

no

Principles of method if other than guideline:

M.J. PRIVAL and V.D. MITCHELL: Analysis of a method for testing azo dyes for mutagenicity in Salmonella typhimurium in the presence of flavine mononucleotide and hamster liver S9, Mutation Research 97: 103-116, 1982
BRUCE N. AMES, JOYCE MCCANN and EDITH YAMASAKI: Methods for Detecting Carcinogens and Mutagens with the Salmonella / Mammalian-Microsome Mutagenicity Test. Mutation Research, 31: 347-364, 1975
DOROTHY M. MARON and BRUCE N. AMES: Revised Method for the Salmonella Mutagenicity Test. Mutation Research, 113: 173-215, 1983

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

EC Number:

260-125-3

EC Name:

N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

Cas Number:

56358-10-2

Molecular formula:

C32H37N5

IUPAC Name:

N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

Test material form:

solid

Details on test material:

Batch number ZD 00883/044. Manufactured Oct 1996
91.3% purity. Impurity is residual solvent Shellsol AB

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

3 experiments were carried out. For each, 5 doses of the test substance are tested with triplicate plating. Dose selection used in further experiments are based on the findings of the first experment.

1st Experiment:
Ames standard plate test, with and without metabolic activation (rat liver S-9 Mix)
Salmonella typhimurium TA98, TA100, TA1535 and TA1537
E.Coli WP2 uvrA
Doses: 5000, 2500, 500, 100, 20 and 0 μg/plate.

2nd Experiment:
Ames pre-incubation test, with and without metabolic activation (rat liver S-9 Mix)
Salmonella typhimurium TA98, TA100, TA1535 and TA1537
E.Coli WP2 uvrA
Doses: 2500, 500, 100, 20, 4 and 0 μg/plate.

3rd Experment:
Prival pre-incubation test, with uninduced hamster liver S-9 Mix
Salmonella typhimurium TA98, TA100, TA1535 and TA1537
Doses: 2500, 500, 100, 20, 4 and 0 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test substance is insoluble in water, so acetone was selected as the vehicle as it has been demontrated to be suitable in bacterial reverse mutation tests for which historical control data are available.

Controls

Untreated negative controls:

yes

Remarks:

Parallel with each experiment, negative controls are carried out in order to check for possible contaminants. The sterility control contains the Agar, S-9Mix, buffer, vehicle. But is WITHOUT the tester stains, and showed no growth.

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle control (acetone) with and without S-9 Mix.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

congo red

other: 2-aminoanthracene (rat liver S-9 & hamster liver S9), benzidine (hamster liver S-9), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (without S-9), 4-nitro-o-phenylendiamine (NOPD) (without S-9), N-ethyl-N'-nitrol-N-nitrosoguanidine (ENNG) (without S-9)

Details on test system and experimental conditions:

Genotypes
Salmonella typhimurium: In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in enhanced sensitivity to some mutagens. Furthermore all strains show a considerably reduced polysaccharide layer (rfa) which leads to an increase in permeability to lipophilic substances. The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. The strains TA 98 and TA 100 carry an R factor plasmid pKM101 and, in addition to having genes resistant to antibiotics, have modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA. This again leads to an increase in sensitivity.
E.Col: WP2 uvrA has an AT base at the primary reversion site is a derivative of E.Coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions. The rate of induced back mutations from Trp- to Trp+ is determined.

Bacteria Storage
The strains are stored at -70 to -80ºC.

Procedure for Growing Cultures
The frozen bacterial cultures were thawed at room temperature and 0,1 ml of this bacterial suspension is inoculated in nutrient broth solutions and incubated in a shaking water bath for 10-12 hours at 37 °C. A density of >10^9 bacteria/ml is reached and the cultures grown overnight are kept in iced water in order to prevent further growth.
0.1ml of these overnight cultures are diluted to 10^-6 and it is 0.1ml of this titer that is added to the test preparations (as described in more detail below)

METABOLIC ACTIVATION SYSTEM
Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system
3 separate experiments were carried out:
Ames standard plate test, with and without metabolic activation (rat liver S-9 Mix)
Ames pre-incubation test, with and without metabolic activation (rat liver S-9 Mix)
Prival pre-incubation test, with uninduced hamster liver S-9 Mix
For azo-dyes and diazo-compounds the modified protocol proposed by Prival and Mitchell is referred to in the OECD guideline No. 471.
This modified protocol differs from the standard plate incorporation assay in five ways:
1. uninduced hamster liver S9 instead of induced rat liver S9 is used
2. the hamster liver S9 mix contains 30% hamster liver extract
3. flavine mononucleotide is added to the S9 mix
4. exogeneous glucose 6-phosphate dehydrogenase, NADH, and four times the standard amount of glucose 6-phosphate is added to the S9 mix
5. a 30 minutes pre-incubation step is used before addition of agar.
These modifications are needed in order to test the mutagenic potential under conditions in which reduction of the compound to its constituent aromatic amines occurs.
Both the Rat Liver S-9 fraction and the Hamster Liver S-9 fraction were prepared at BASF and preparation information is available in the referenced report.

DESCRIPTION OF THE TEST PROCEDURE
The study included the following tests:
3 experiments were carried out, for each sample type described (control or test), 3 plates of each were prepared (i.e triplicate). The results reported are the mean revertant count values.

Ames standard plate test, with and without metabolic activation (rat liver S-9 Mix)
Doses: 5000, 2500, 500, 100, 20 and 0 μg/plate.

Ames pre-incubation test, with and without metabolic activation (rat liver S-9 Mix)
Doses: 2500, 500, 100, 20, 4 and 0 μg/plate.

Prival pre-incubation test, with uninduced hamster liver S-9 Mix
Doses: 2500, 500, 100, 20, 4 and 0 μg/plate.

The procedures for each were as follows:

Ames standard plate test:

Test tubes containing 2ml of agar were kept in a water bath at 45C and the remaining components added in the following order
0.1ml test solution (or vehicle)
0.1ml fresh bacterial culture (dilution 10-6)
0.5ml S-9 mix (in tests with metabolic activation OR 0.5ml phosphate buffer (in tests without metabolic activation.
After mixing the samples are poured onto agar plates within approx. 30 seconds. The agar compositions for both Salmonella are E.Coli are documented.

After preparation, the plates were incubated at 37 °C for 48 -72 hours in the dark, after which the bacterial colonies are counted (his+ revertants for Salmonella or trp+ revertants for E.Coli)

Ames pre-incubation test.
The following components are placed together and incubated at 37C for 20 minutes using a shaker:
0.1ml test solution (or vehicle)
0.1ml fresh bacterial culture (dilution 10-6)
0.5ml S-9 mix. (rat liver)
After incubation, 2ml of agar is added and mixed. The samples are then poured onto agar plates within 30 seconds and after incubation at 37C in the dark for 48-72 hours the colonies are counted.

Prival pre-incubation test.
The following components are placed together and incubated at 30C for 30 minutes using a shaker:
0.1ml test solution (or vehicle)
0.1ml fresh bacterial culture (dilution 10-6)
0.5ml S-9 mix (hamster liver)
After incubation, 2ml of agar is added and mixed. The samples are then poured onto agar plates within 30 seconds and after incubation at 37C in the dark for 48-72 hours the colonies are counted.


Control Groups Used in the Tests
Strain-specific negative (solvent) controls, were included in each test.
Positive controls were also used to check the mutability of the bacteria and the activity of the S-9 mix. The results for the positive controls are also reported in the results tables. The control used was selected depending on the strain and/or the method used. The controls used were as follows:
-with rat liver S-9 Mix
2-aminoanthracene (2-AA)
2.5µg/plate dissolved in DMSO, for strains TA 1535, TA 100, TA 1537, TA 98
60µg/plate dissolved in DMSO, for E.Coli

-with hamster liver S-9 Mix
2-aminoanthracene (2-AA) 10µg/plate dissolved in DMSO, for strains TA 1535, TA 100, TA 1537, TA 98
Congo Red: 0.3µmol/plate dissolved in DMSO for TA 98
Benzidine: 0.3µmol/plate dissolved in DMSO for TA 98

-Without S-9 Mix
N-methyl-N'-nitrol-N-nitrosoguanidine (MNNG) 5µg/plate dissolved in DMSO for TA 1535, TA 100
4-nitro-o-phenylendiamine (NOPD): 10µg/plate dissolved in DMSO for TA-98
9-aminoacridine (AAC): 100µg/plate dissolved in DMSO for TA1537
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 10µg/plate dissolved in DMSO for E.Coli

In addition, historic negative control data and historic positive control data were also reviewed to confirm the suitability of the vehicle and the methodology used.

Titer: The titer is generally determined only in the experimental parts with S-9 mix for the negative controls and for the 2 highest doses in all experiments.

Toxicity (if detected) was recorded for all test groups. This is when there is a decrease in the number of revertants, a clearing or dimunition of the background lawn, or a reduction in the titer.

Solubiity: Precipitation of the test material was recorded

Rationale for test conditions:

In accordance with the test guidelines.

Evaluation criteria:

The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting.

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) controls was within the normal range of historical control data for each tester strain.
-the sterility controls revealed no indication of bacterial contamination (i.e no growth should be seen)
- The postive control (with and without activation) induced a significant increase in the number of revertant colonies within the range of historical control data.
-The titer of viable bacteria was >10^9

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related and reproducibe increase in the number of revertants occurred (i.e a doubling of the spontaneous mutation rate in at least one tester strain with or without S-9 mix)

Criteria for a Negative Response:
A test article was considered non-mutagenic if:
- the total number of revertants for all tester strains were within the historical negative control range under all experimental conditions in 2 experiments carried out independently of each other.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls. For each sample type, 3 plates were prepared. The values given in the results table are the mean values from the triplicate results.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Untreated, negative (vehicle) and positive controls were run concurrently. The mean values of revertant colony numbers in the negative, and postive control plates were within the historical control range in all strains. The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and withuoth matabolic activastion. The study was considered to be valid.

Any other information on results incl. tables

STANDARD PLATE TEST RESULTS

 

Concentration (μg/plate)	Mean Revertants per plate / Mutation Factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA-1535		TA-100		TA-1537		TA-98		WP2 uvra
		Without S-9	With S-9	Without S-9	With S-9	Without S-9	With S-9	Without S-9	With S-9	Without S-9	With S-9
Acetone control	Mean	22	20	117	123	10	12	31	41	33	38
	MF	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
2-AA 2.5µg	Mean		137		1760		183		1484
	MF		6.8		14.3		15.7		36.2
2-AA 60µg	Mean										216
	MF										5.7
MNNG 5µg	Mean	1286		1349
	MF	58.5		11.5
AAC 100µg	Mean					781
	MF					75.5
NOPD 10µg	Mean							1206
	MF							38.5
ENNG 10µg	Mean									1023
	MF									31.3
20µg	Mean	20	18	113	103	9	12	28	36	31	32
	MF	0.9	0.9	1.0	0.8	0.8	1.0	0.9	0.9	0.9	0.8
100µg	Mean	19 P	16 P	115 P	121 P	8 P	9 P	26 P	36 P	34 P	32 P
	MF	0.9	0.8	1.0	1.0	0.8	0.8	0.8	0.9	1.0	0.8
500µg	Mean	13 P	16P	97 P	128 P	7 P	8 P	27 P	31 P	30 P	37 P
	MF	0.6	0.8	0.8	1.0	0.7	0.7	0.9	0.8	0.9	1.0
2500µg	Mean	8 P	13 P	165 P	134 P	6 P	8 P	27 P	29 P	30 P	43 P
	MF	0.4	0.7	1.4	1.1	0.5	0.7	0.9	0.7	0.9	1.2
5000µg	Mean	6 P	10 P	141 P	154 P	4 P	7 P	18 P	22 P	34 P	42 P
	MF	0.3	0.5	1.2	1.3	0.4	0.6	0.6	0.5	1.0	1.1

P = Precipitation

 

 

AMES PREINCUBATION TEST

Concentration (μg/plate)	Mean Revertants per plate / Mutation Factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA-1535		TA-100		TA-1537		TA-98		WP2 uvra
		Without S-9	With S-9	Without S-9	With S-9	Without S-9	With S-9	Without S-9	With S-9	Without S-9	With S-9
Acetone control	Mean	20	20	118	130	10	9	31	39	32	42
	MF	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
2-AA 2.5µg	Mean		181		1256		186		943
	MF		9.1		9.6		19.9		24.0
2-AA 60µg	Mean										203
	MF										4.8
MNNG 5µg	Mean	1024		1345
	MF	52.1		11.4
AAC 100µg	Mean					419
	MF					43.3
NOPD 10µg	Mean							1240
	MF							39.6
ENNG 10µg	Mean									833
	MF									26.0
4µg	Mean	17	15	109	140	10	11	28	38	28	33
	MF	0.9	0.8	0.9	1.1	1.1	1.1	0.9	1.0	0.9	0.8
20µg	Mean	18	15	103	123	9	9	28	35	32	30
	MF	0.9	0.8	0.9	0.9	0.9	1.0	0.9	0.9	1.0	0.7
100µg	Mean	15 P	16P	101 P	129 P	10 P	9 P	25 P	38 P	30 P	30 P
	MF	0.8	0.8	0.9	1.0	1.0	0.9	0.8	1.0	0.9	0.7
500µg	Mean	17 P	15	100 P	109 P	8 P	10 P	25 P	26 P	27 P	30 P
	MF	0.9	0.8	0.8	0.8	0.8	1.0	0.8	0.7	0.8	0.7
2500µg	Mean	11 P	11	97 P	105 P	7 P	9 P	22 P	25 P	27 P	26 P
	MF	0.5	0.5	0.8	0.8	0.7	0.9	0.7	0.6	0.9	0.6

P = Precipitation

 

 

PRIVAL PREINCUBATION TEST

		Salmonella typhimurium tester strains
Concentration (μg/plate)	Mean Revertants per plate / Mutation Factor (MF)	TA-1535 With S-9	TA-100 With S-9	TA-1537 With S-9	TA-98 With S-9
Acetone control	Mean	19	118	10	42
	MF	1.0	1.0	1.0	1.0
2-AA 10µg	Mean	151	922	247	606
	MF	8.1	7.8	23.9	14.4
CONGO RED 210µg	Mean				168
	MF				4.0
BENZIDINE 55µg	Mean				909
	MF				21.7
4µg	Mean	17	108	8	39
	MF	0.9	0.9	0.8	0.9
20µg	Mean	15	106	6	40
	MF	0.8	0.9	0.6	1.0
100µg	Mean	14 P	90 P	4 P	40 P
	MF	0.8	0.8	0.4	0.9
500µg	Mean	14 P	74 P	5 P	42 P
	MF	0.7	0.6	0.5	1.0
2500µg	Mean	11 P	123 P	9 P	45 P
	MF	0.6	1.0	0.9	1.1

P = Precipitation

Applicant's summary and conclusion

Conclusions:

The test item Solvent Red 500 was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, in a Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system.
An increase in the number of revertants was not observed in either of the Ames test or in the Prival test, with or without the addition of metabolizing systems.
Therefore Solvent Red 500 is not mutagenic in the bacterial reverse mutation assay.
 

Executive summary:

The test item Solvent Red 500 was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system. 3 separate experiments were carried out,

Standard Plate test (with a dose range of 20 -5000µg/Plate), with and without metabolic activation (rat liver S-9 mix)

Ames pre-incubation test (with a dose range of 20 -2500µg/Plate),with and without metabolic activation (rat liver S-9 mix)

Prival pre-incubation test (with a dose range of 20 -2500µg/Plate), with metabolic activation (hamster liver S-9 mix)

Precipitation of the test substance was observed from about 100µg/plate and higher.

A slight decrease in the number of revertants was occassionally observed depending on the strain and test conditions from about 500µg/plate and higher.

An increase in the number of revertants was not observed in either of the Ames test with or without the addition of metabolizing system

or in the Prival test with metabolic activation.

Therefore Solvent Red 500 is not mutagenic in the bacterial reverse mutation assay.