N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)azo]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:

2016 -03-08 till 2016-04-05

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:

2016

Report date:

2016

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:

Pigment Additiv FGR

IUPAC Name:

Pigment Additiv FGR

Constituent 2

Reference substance name:

N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

EC Number:

225-691-8

EC Name:

N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Cas Number:

5012-29-3

Molecular formula:

C25H18Cl3N3O3

IUPAC Name:

N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)diazenyl]-2-naphthamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: Pigment Additiv FGR
Batch: FB3212.013
CAS-No.: 5012-29-3
EINECS / EC-No.: 225-691-8
Purity: 94.51% (w/w), dose calculation was not be adjusted to purity
Appearance: Brown powder
Expiry Date: 10.05.2020 (Statement of producer)
Storage Conditions: At room temperature
Stability in Solvent: > 72 h in DMSO at room temperature
> 72 h in 1,2-propylene glycol at room temperature
Certificate of Analysis: AZ 577/Toxd2, dated 11. December 2015

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 (experiment I) and non-induced hamster liver (experiment II)

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
3, 10, 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Best suitable solvent

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
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, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp. I strain WP2 uvrA with S9mix

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
- Precipitation:The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: In experiment II, the data in the negative control of strains TA 1535 with S9 mix and TA1537 with S9 mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabol¬ic activation in the first experiment. Only in experiment I minor toxic effects were observed in strain WP2 uvrA with S9 mix at 5000 µg/plate.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tabellen

Table1            Summary of Experiment I

Study Name: 1755506	Study Code: Envigo 1755506
Experiment: 1755506 VV Plate	Date Plated: 08/03/2016
Assay Conditions:	Date Counted: 11/03/2016

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			16 ± 1	9 ± 6	25 ± 7	186 ± 15	51 ± 4
	Untreated			13 ± 4	8 ± 2	22 ± 7	194 ± 15	51 ± 3
	Pigment-	3 µg		11 ± 2	12 ± 6	29 ± 3	191 ± 7	47 ± 12
	Additiv FGR	10 µg		13 ± 3	12 ± 4	31 ± 4	197 ± 17	50 ± 16
		33 µg		14 ± 7	9 ± 2	22 ± 2	165 ± 8	45 ± 12
		100 µg		15 ± 2	11 ± 3	30 ± 9	178 ± 13	40 ± 10
		333 µg		16 ± 5^P	10 ± 2^P	34 ± 7^P	178 ± 2^P	49 ± 9^P
		1000 µg		11 ± 3^P	10 ± 1^P	26 ± 6^P	199 ± 15^P	48 ± 7^P
		2500 µg		11 ± 3^P	7 ± 1^P	24 ± 6^P	185 ± 12^P	45 ± 4^P
		5000 µg		13 ± 3^P	11 ± 2^P	32 ± 10^P	199 ± 17^P	46 ± 4^P
	NaN3	10 µg		1205 ± 83			2418 ± 75
	4-NOPD	10 µg				407 ± 22
	4-NOPD	50 µg			83 ± 14
	MMS	2.0 µL						1056 ± 53
With Activation	DMSO			12 ± 4	16 ± 3	39 ± 3	196 ± 33	51 ± 14
	Untreated			14 ± 6	11 ± 3	39 ± 14	175 ± 26	61 ± 10
	Pigment-	3 µg		11 ± 6	15 ± 2	39 ± 8	203 ± 34	55 ± 1
	Additiv FGR	10 µg		12 ± 3	13 ± 4	40 ± 12	212 ± 53	49 ± 8
		33 µg		12 ± 5	13 ± 2	43 ± 14	210 ± 51	54 ± 14
		100 µg		14 ± 3	15 ± 1	32 ± 4	193 ± 14	56 ± 4
		333 µg		12 ± 4^P	13 ± 3^P	31 ± 11^P	178 ± 23^P	50 ± 3^P
		1000 µg		14 ± 3^P	13 ± 1^P	40 ± 3^P	176 ± 7^P	57 ± 14^P
		2500 µg		9 ± 1^P	15 ± 2^P	31 ± 7^P	186 ± 24^P	64 ± 2^P
		5000 µg		11 ± 5^P	9 ± 4^P	32 ± 5^P	192 ± 19^P	22 ± 5^P M
	2-AA	2.5 µg		539 ± 19	92 ± 13	4980 ± 327	3152 ± 55
	2-AA	10.0 µg						476 ± 25

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

 

 

 

Table2            Summary of Experiment II

Study Name: 1755506	Study Code: Envigo 1755506
Experiment: 1755506 HV2 Pre	Date Plated: 31/03/2016
Assay Conditions:	Date Counted: 05/04/2016

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			12 ± 2	11 ± 3	28 ± 10	151 ± 15	38 ± 6
	Untreated			13 ± 2	8 ± 2	26 ± 9	182 ± 28	51 ± 4
	Pigment-	3 µg		12 ± 1	11 ± 2	32 ± 8	141 ± 4	42 ± 4
	Additiv FGR	10 µg		11 ± 4	13 ± 2	29 ± 4	148 ± 15	44 ± 1
		33 µg		12 ± 5	10 ± 1	27 ± 5	141 ± 17	42 ± 2
		100 µg		11 ± 2	11 ± 3	24 ± 4	138 ± 21	38 ± 6
		333 µg		12 ± 1^P	11 ± 1^P	23 ± 6^P	153 ± 17^P	41 ± 1^P
		1000 µg		14 ± 2^P	10 ± 2^P	25 ± 11^P	158 ± 12^P	37 ± 4^P
		2500 µg		11 ± 2^P	8 ± 1^P	20 ± 3^P	153 ± 17^P	39 ± 8^P
		5000 µg		12 ± 4^P	6 ± 1^P	21 ± 4^P	116 ± 17^P	43 ± 4^P
	NaN3	10 µg		1211 ± 42			2161 ± 107
	4-NOPD	10 µg				487 ± 52
	4-NOPD	50 µg			80 ± 11
	MMS	2 µL						981 ± 56
With Activation	DMSO			21 ± 4	24 ± 6	51 ± 7	112 ± 9	66 ± 15
	Untreated			27 ± 8	30 ± 4	53 ± 5	175 ± 10	72 ± 11
	Pigment-	3 µg		25 ± 7	27 ± 4	56 ± 10	109 ± 3	60 ± 7
	Additiv FGR	10 µg		27 ± 7	18 ± 2	54 ± 7	127 ± 6	68 ± 11
		33 µg		29 ± 9	18 ± 4	45 ± 8	132 ± 8	68 ± 4
		100 µg		30 ± 4	23 ± 3	58 ± 13	138 ± 12	67 ± 6
		333 µg		27 ± 4^P	29 ± 3^P M	59 ± 7^P	134 ± 18^P	61 ± 6^P
		1000 µg		18 ± 4^P	22 ± 3^P M	55 ± 2^P	110 ± 4^P	65 ± 11^P
		2500 µg		16 ± 1^P	21 ± 5^P M	52 ± 10^P	101 ± 7^P	63 ± 5^P
		5000 µg		15 ± 2^P	19 ± 5^P M	31 ± 4^P	94 ± 1^P	48 ± 4^P
	2-AA	2.5 µg					1592 ± 269
	2-AA	2.5 µg		328 ± 45	149 ± 7
	2-AA	10 µg						953 ± 63
	Congo red	500 µg				434 ± 119

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD Congo red MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine Congo red methyl methane sulfonate	P M	Precipitate Manual count

 

 

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 with and without rat and hamster S9.

Executive summary:

Discussion von Bericht

This study was performed to investigate the potential of Pigment-Additiv FGR to induce gene muta­tions according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

 

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations in both experiments:

3; 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. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate. 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.

Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain WP2 uvrA with S9 mix at 5000 µg/plate in experiment I. No toxic effects occurred in the remaining strains with and without metabolic activation in experiment I and II.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pigment-Additiv FGR at any concentration level, neither in the presence nor absence of metabolic activation (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.

In experiment II, the data in the negative control of strains TA 1535 with S9 mix and TA1537 with S9 mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.