4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide

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:

2017-07-19 till 2017-10-02

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:

4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide

EC Number:

229-104-6

EC Name:

4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide

Cas Number:

6410-38-4

Molecular formula:

C24H17Cl2N3O3

IUPAC Name:

4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Name of test material (as cited in study report): Part of several studies carried out by Envigo (see study record for report name); Pigment Red 9;
Analytical purity: 99.7%

Specific details on test material used for the study:

IIdentification: Pigment Red 9
Batch: JPIB000140ger
CAS No.: 6410-38-4
EINECS / EC No.: 229-104-6

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 II9

Test concentrations with justification for top dose:

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

Vehicle / solvent:

Solvent used: DMSO
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 30°C
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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strains TA 1535, TA 1537 and TA 98 (experiment I)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment II. The undissolved particles had no influence on the data recording.
Other confounding effects: 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, except strain TA 98 in experiment II in the absence and presence of metabolic activation. The tester strain TA 98 showed an extensive bacterial background growth indicated by a more dense background lawn. Thus, in experiment II all plates incubated with strain TA 98 were scored manually. The revertant rates of both negative control groups each were within our laboratory’s historical negative control range for strain TA 98 and, therefore, this observation has to be regarded to have no detrimental impact on the validity and outcome of the study.

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in several strains with and without metabolic activation in experiment I (plate incubation method) at the top dose of 5000 µg/plate.

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: 1802502	Study Code: Envigo 1802502
Experiment: 1802502 VV Plate	Date Plated: 19.09.2017
Assay Conditions:	Date Counted: 22.09.2017

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 100Revertant Colony Counts (Mean ± SD)	WP2 uvrA Revertant Colony Counts (Mean ± SD)
Without	DMSO		14 ± 2	8 ± 2	28 ± 8	129 ± 16	29 ± 3
	Untreated		14 ± 2	12 ± 2	22 ± 2	156 ± 9	43 ± 5
Activation	Pigment	3 µg	15 ± 5	9 ± 4	26 ± 9	129 ± 9	31 ± 4
	Red 9	10 µg	12 ± 5	10 ± 4	28 ± 11	117 ± 8	41 ± 1
		33 µg	15 ± 4	11 ± 4	24 ± 2	133 ± 8	31 ± 5
		100 µg	12 ± 3	11 ± 5	24 ± 8	117 ± 16	40 ± 7
		333 µg	11 ± 4	9 ± 3	19 ± 3	120 ± 3	39 ± 11
		1000 µg	11 ± 3^P	7 ± 2^P	27 ± 9^P	113 ± 4^P	28 ± 7^P
		2500 µg	13 ± 6^P	7 ± 2^P	24 ± 7^P	118 ± 12^P	42 ± 7^P
		5000 µg	7 ± 1^P M	4 ± 2^P M	12 ± 2^P M	99 ± 8^P M	18 ± 3^P M
	NaN3	10 µg	1115 ± 98			1923 ± 66
	4-NOPD	10 µg			375 ± 33
	4-NOPD	50 µg		79 ± 3
	MMS	2.0 µL					1003 ± 107
With	DMSO		15 ± 7	13 ± 4	40 ± 12	105 ± 5	47 ± 2
Activation	Untreated		17 ± 4	12 ± 5	28 ± 2	132 ± 6	57 ± 11
	Pigment	3 µg	11 ± 1	17 ± 2	36 ± 4	107 ± 11	38 ± 2
	Red 9	10 µg	15 ± 7	16 ± 2	32 ± 2	104 ± 12	51 ± 4
		33 µg	13 ± 4	11 ± 3	35 ± 9	111 ± 18	47 ± 4
		100 µg	11 ± 1	11 ± 1	35 ± 6	111 ± 20	43 ± 4
		333 µg	13 ± 4	12 ± 6	40 ± 9	103 ± 19	45 ± 7
		1000 µg	14 ± 4^P	8 ± 3^P	29 ± 8^P	100 ± 20^P	39 ± 4^P
		2500 µg	10 ± 2^P M	9 ± 3^P	28 ± 3^P	104 ± 6^P	42 ± 9^P
		5000 µg	4 ± 1^P M	3 ± 1^P M	14 ± 3^P M	67 ± 5^P M	28 ± 5^P M
	2-AA	2.5 µg	387 ± 22	130 ± 14	3164 ± 115	3389 ± 140
	2-AA	10.0 µg					441 ± 111

Summary of Experiment II

Study Name: 1802502	Study Code: Envigo 1802502
Experiment: 1802502 HV2 Pre	Date Plated: 26.09.2017
Assay Conditions:	Date Counted: 02.10.2017

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	DMSO		9 ± 2	9 ± 2	15 ± 2^B M	151 ± 4	40 ± 4
Activation	Untreated		10 ± 3	11 ± 4	14 ± 2^B M	187 ± 15	34 ± 3
	Pigment	10 µg	7 ± 1	9 ± 4	19 ± 4^B M	163 ± 30	37 ± 8
	Red 9	33 µg	10 ± 1	10 ± 2	19 ± 2^B M	166 ± 5	27 ± 3
		100 µg	7 ± 2	10 ± 1	16 ± 1^B M	158 ± 10	32 ± 4
		333 µg	7 ± 2^P	9 ± 3^P	17 ± 3^P B M	152 ± 14^P	34 ± 2^P
		1000 µg	9 ± 3^P	8 ± 2^P	17 ± 0^P B M	157 ± 4^P	33 ± 5^P
		2500 µg	10 ± 2^P M	8 ± 2^P M	13 ± 3^P B M	140 ± 5^P M	29 ± 6^P M
		5000 µg	8 ± 2^P M	7 ± 1^P M	14 ± 4^P B M	132 ± 5^P M	29 ± 3^P M
	NaN3	10 µg	1357 ± 116			2093 ± 303
	4-NOPD	10 µg			395 ± 21^B M
	4-NOPD	50 µg		91 ± 5
	MMS	2 µL					1135 ± 38
With	DMSO		12 ± 4	15 ± 4	16 ± 1^B M	134 ± 3	35 ± 7
Activation	Untreated		11 ± 4	18 ± 2	12 ± 1^B M	140 ± 9	40 ± 3
	Pigment	10 µg	9 ± 2	14 ± 6	18 ± 3^B M	129 ± 9	38 ± 7
	Red 9	33 µg	10 ± 4	12 ± 5	13 ± 3^B M	143 ± 10	35 ± 7
		100 µg	13 ± 2	21 ± 1	13 ± 2^B M	151 ± 13	40 ± 5
		333 µg	8 ± 3^P	18 ± 6^P	17 ± 2^P B M	130 ± 11^P	38 ± 9^P
		1000 µg	11 ± 6^P	21 ± 2^P	17 ± 3^P B M	156 ± 9^P	38 ± 8^P
		2500 µg	7 ± 2^P M	14 ± 2^P M	13 ± 1^P B M	147 ± 4^P M	26 ± 1^P M
		5000 µg	10 ± 1^P M	20 ± 3^P M	11 ± 1^P B M	120 ± 7^P M	22 ± 2^M P
	2-AA	2.5 µg				2218 ± 122
	2-AA	2.5 µg	378 ± 9	238 ± 21
	2-AA	10 µg					777 ± 45
	Congo red	500 µg			2027 ± 161^B M

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

B:  Extensive bacterial colny growth

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 not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Pigment Red 9 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item Pigment Red 9 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:

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

Experiment II:                           10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment II. 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, except strain TA 98 in experiment II in the absence and presence of metabolic activation. The tester strain TA 98 showed an extensive bacterial background growth indicated by a more dense background lawn. Thus, in experiment II all plates incubated with strain TA 98 were scored manually. The revertant rates of both negative control groups each were within our laboratory’s historical negative control range for strain TA 98 and, therefore, this observation has to be regarded to have no detrimental impact on the validity and outcome of the study.

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I without S9 mix	Experiment I with S9 mix	Experiment II without S9 mix	Experiment II with S9 mix
TA 1535	/	5000	/	/
TA 1537	/	5000	/	/
TA 98	5000	5000	/	/
TA 100	/	/	/	/
WP2 uvrA	/	/	/	/

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pigment Red 9 at any concentration level, neither in the presence nor absence of metabolic activation (rat or hamster S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

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