1-[(4-methoxy-2-nitrophenyl)azo]-2-naphthol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-02 till 2017-05-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-[(4-methoxy-2-nitrophenyl)azo]-2-naphthol

EC Number:

256-458-9

EC Name:

1-[(4-methoxy-2-nitrophenyl)azo]-2-naphthol

Cas Number:

49744-28-7

Molecular formula:

C17H13N3O4

IUPAC Name:

1-[(4-methoxy-2-nitrophenyl)diazenyl]-2-naphthol

Test material form:

solid

Specific details on test material used for the study:

IIdentification: 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol
CAS No.: 49744-28-7
EINECS / EC No.: 256-458-8
Batch: 3.April 2010 g
Purity: 99.2% (w/w) including isomer
Physical State / Appearance: Dark red powder
Expiry Date: 25 January 2027 (Statement of sponsor)
Storage Conditions: At room temperature
Certificate of Analysis: AZ 1070/Toxd1, dated 25 January 2017
Stability in Solvent: Not indicated by the Sponsor

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9 (experiment I) non- induced hamster liver S9 (experiment II)

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
All strains without S9 mix: 33; 100; 333; 1000; 2500; and 5000 µg/plate
All strains with S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Solvent used: DMF
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 30 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

strain TA 98 with non-induced hamster liver S9

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not soluble at 50 mg/mL
Precipitation: The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate with and without S9 mix in experiment I and in experiment II from 1000 to 5000 µg/plate without S9 mix and from 333 to 5000 µg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate with and without S9 mix and in experiment II from 2500 to 5000 µg/plate without S9 mix and from 1000 to 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviation
ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Remarks on result:

other: reverse mutation assay migrated from the field Test System

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1839700	Study Code: Envigo 1839700
Experiment: 1839700 VV Plate	Date Plated: 02.05.2017
Assay Conditions:	Date Counted: 08.05.2017

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	DMF		11 ± 5	8 ± 3	20 ± 6	151 ± 25	35 ± 4
Activation	Untreated		8 ± 4	9 ± 3	22 ± 3	160 ± 21	40 ± 6
	1-((4-Methoxy-2-	3 µg	8 ± 2	9 ± 4	17 ± 4	149 ± 6	38 ± 2
	nitrophenyl)azo)-	10 µg	10 ± 1	12 ± 3	22 ± 6	144 ± 16	45 ± 9
	2-naphthol	33 µg	11 ± 1	12 ± 3	25 ± 2	145 ± 22	36 ± 6
		100 µg	8 ± 4	10 ± 4	23 ± 8	158 ± 16	45 ± 8
		333 µg	8 ± 2	10 ± 2	19 ± 3	154 ± 12	39 ± 5
		1000 µg	11 ± 4	13 ± 4	19 ± 8	135 ± 9	33 ± 8
		2500 µg	10 ± 5^P	13 ± 1^P	19 ± 2^P	157 ± 8^P	36 ± 5^P
		5000 µg	10 ± 1^P	11 ± 1^P	22 ± 6^P M	150 ± 26^P	41 ± 11^P
	NaN3	10 µg	1164 ± 122			2017 ± 102
	4-NOPD	10 µg			262 ± 7
	4-NOPD	50 µg		78 ± 5
	MMS	2.0 µL					1030 ± 98
With	DMF		9 ± 2	15 ± 3	24 ± 4	134 ± 16	51 ± 4
Activation	Untreated		10 ± 5	13 ± 4	33 ± 9	157 ± 18	53 ± 9
	1-((4-Methoxy-2-	3 µg	9 ± 1	14 ± 2	30 ± 2	147 ± 8	52 ± 6
	nitrophenyl)azo)-	10 µg	7 ± 2	17 ± 5	26 ± 5	159 ± 14	50 ± 10
	2-naphthol	33 µg	11 ± 1	16 ± 4	30 ± 2	161 ± 14	58 ± 7
		100 µg	7 ± 1	16 ± 4	36 ± 7	151 ± 10	53 ± 5
		333 µg	10 ± 4	19 ± 3	34 ± 7	173 ± 15	52 ± 4
		1000 µg	9 ± 3	20 ± 2	30 ± 5	156 ± 28	46 ± 9
		2500 µg	12 ± 2^P	19 ± 6^P	32 ± 5^P	164 ± 8^P	53 ± 7^P
		5000 µg	11 ± 1^P	16 ± 4^P M	33 ± 3^P M	140 ± 14^P M	50 ± 13^P
	2-AA	2.5 µg	409 ± 13	171 ± 4	3450 ± 1007	4360 ± 355
	2-AA	10.0 µg					327 ± 57

Summary of Experiment II

Study Name: 1839700	Study Code: Envigo 1839700
Experiment: 1839700 HV2 Pre	Date Plated: 11.05.2017
Assay Conditions:	Date Counted: 16.05.2017

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	DMF		9 ± 3	9 ± 2	30 ± 7	147 ± 8	33 ± 7
Activation	Untreated		9 ± 3	9 ± 2	32 ± 0	193 ± 19	32 ± 1
	1-((4-Methoxy-2-	33 µg	11 ± 3	10 ± 3	29 ± 6	137 ± 6	44 ± 7
	nitrophenyl)azo)-	100 µg	7 ± 2	11 ± 1	24 ± 5	153 ± 10	42 ± 12
	2-naphthol	333 µg	8 ± 2	13 ± 3	30 ± 3	148 ± 7	39 ± 10
		1000 µg	8 ± 2	8 ± 2	22 ± 7	149 ± 3	34 ± 18
		2500 µg	8 ± 2^P	8 ± 3^P	27 ± 6^P	152 ± 13^P	26 ± 4^P
		5000 µg	7 ± 2^P	11 ± 3^P	19 ± 4^P M	143 ± 1^P M	23 ± 5^P M
	NaN3	10 µg	1103 ± 46			2039 ± 76
	4-NOPD	10 µg			267 ± 5
	4-NOPD	50 µg		79 ± 8
	MMS	2.0 µL					1094 ± 36
With	DMF		10 ± 1	23 ± 7	41 ± 1	138 ± 4	43 ± 6
Activation	Untreated		7 ± 2	21 ± 9	38 ± 3	126 ± 27	48 ± 6
	1-((4-Methoxy-2-	3 µg	10 ± 2	28 ± 3	48 ± 6	126 ± 10	44 ± 3
	nitrophenyl)azo)-	10 µg	12 ± 8	31 ± 10	52 ± 10	148 ± 12	48 ± 4
	2-naphthol	33 µg	12 ± 1	23 ± 2	61 ± 15	157 ± 7	50 ± 11
		100 µg	15 ± 2	23 ± 3	81 ± 6	170 ± 14	50 ± 21
		333 µg	20 ± 4	25 ± 5	124 ± 17	205 ± 46	49 ± 16
		1000 µg	17 ± 8^P	24 ± 7^P	104 ± 14^P	219 ± 11^P	54 ± 3^P
		2500 µg	10 ± 2^P	21 ± 6^P	85 ± 12^P M	176 ± 10^P M	41 ± 7^P M
		5000 µg	16 ± 3^P M	25 ± 6^P M	78 ± 8^P M	196 ± 11^P M	36 ± 6^P M
	2-AA	2.5 µg	372 ± 22	269 ± 9		2382 ± 92
	2-AA	10.0 µg					800 ± 28
	Congo red				440 ± 48

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

R:  Reduced Background growth

D:  Densely Colored Plate

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 in the presence of non-induced hamster liver S9 mix.
Therefore, 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and Experiment II was performed with non-induced hamster liver S9 mix. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

All strains without S9 mix:             33; 100; 333; 1000; 2500; and 5000 µg/plate

All strains with S9 mix:                  3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate with and without S9 mix in experiment I and in experiment II from 1000 to 5000 µg/plate without S9 mix and from 333 to 5000 µg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate with and without S9 mix and in experiment II from 2500 to 5000 µg/plate without S9 mix and from 1000 to 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

In experiment II an increase in revertant colony numbers was observed following treatment with 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol in strain TA 98 in the presence of metabolic activation (non-induced hamster liver S9 mix).The number of colonies exceeded the threshold of twice the number of the corresponding solvent control from 333 to 2500 µg/plate. The induction factor decreased with the increasing precipitation of the test substance and at a concentration of 5000µg/plate the threshold of twice the number of the corresponding solvent control was not reached. This effect may be based on the observed precipitation of the test item which causes a minor bio-availability of the test substance or an overlapping toxic effect.

A minor increase in revertant colony numbers, not reaching the threshold of thrice the number of the corresponding solvent control, was observed in strain TA 1535in the presence of metabolic activation (S9 mix).

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.