2-[(8-amino-7-{[4-substituted-2-sulfonatophenyl]diazenyl}-1-hydroxy-3,6-disulfonaphthalen-2-yl)diazenyl]-4-[(4-chloro-6-{[3-(substituted)phenyl](ethyl)amino}-heteromonocycl-2-yl)amino]arylsulfonic acid, potassium and sodium salts

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:

2009-11-30 till 2009-12-14

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:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: "Kanpoan No. 287 -- Environment Protection Agency" "Eisei No. 127 -- Ministry of Health &Welfarew "Heisei 09110131 Kikyoku No. 2 -- Ministry of International Trade & Industry"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): FAT 40849/A TE
- Substance type: reactive dyestuff for cellulose fibers
- Physical state: blue powder
- Analytical purity: 76.9% of all colored components
- Lot/batch No.: TZ 5463 / BOP 03-09
- Expiration date of the lot/batch: 2014-06-30
- Storage condition of test material: At room temperature at about 20 °C

Method

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 applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

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

Vehicle / solvent:

deionised water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 1 hour
- Exposure duration: approx 72h



NUMBER OF REPLICATIONS: 3 plates



DETERMINATION OF CYTOTOXICITY
- Method:reduction in the number of revertants (below the indication
factor of 0.5)

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: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

(not relevant)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar from 1000 Lg/plate up to 5000 Lg/plate in both experiments. No precipitation was observed on the incubated agar plates. The undissolved particles had no influence on the data recording


RANGE-FINDING/SCREENING STUDIES:The pre-experiment is reported as experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA:the spontaneous reversion rates in the negative and solvent control are in the range of
our historical data

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed normal background growth up to 5000 Lg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication
factor of 0.5), occurred in almost all strains the with and without metabolic activation. A
minor reduction in the number of revertants (below the indication factor of 0.5), occurred in
strain TA 1537 and TA 98.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results Pre-Experiment and Experiment I

 

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	Deionised water		12 ± 2	15 ± 0	25 ± 2	130 ± 16	44 ± 9
	Untreated		11 ± 2	15 ± 1	25 ± 6	127 ± 17	44 ± 12
	FAT 40849/A TE	3μg	11 ± 2	8 ± 3	28 ± 4	127 ± 1	50 ± 4
		10μg	12 ± 2	9 ± 4	23 ± 2	121 ± 12	48 ± 9
		33μg	13 ± 1	12 ± 0	22 ± 6	121 ± 6	46 ± 2
		100μg	9 ± 1^U M	9 ± 2	24 ± 9	134 ± 1	50 ± 1
		333μg	7 ± 2^U M	6 ± 3	26 ± 5	116 ± 18	50 ± 2
		1000μg	9 ± 2^U M	5 ± 2	29 ± 1	109 ± 2	51 ± 1
		2500μg	12 ± 3^D M U	3 ± 3^D M	16 ±^D M	113 ± 8^D M	43 ± 4^D M
		5000μg	8 ± 3^D M U	6 ± 2^D M	17 ± 4^D M	114 ± 8^D M	47 ± 3^D M
	NaN3	10μg	1884 ± 28			2059 ± 181
	4-NOPD	10μg			220 ± 2
	4-NOPD	50μg		339 ± 2
	MMS	3.0μL					1161 ± 26
With Activation	Deionised water		14 ± 1	12 ± 2	34 ± 7	140 ± 7	60 ± 2
	Untreated		15 ± 1	15 ± 5	38 ± 5	150 ± 10	63 ± 14
	FAT 40849/A TE	3μg	16 ± 4^U M	13 ± 1	35 ± 6	142 ± 18	62 ± 6
		10μg	11 ± 3^U M	10 ± 2	35 ± 6	151 ± 18	58 ± 7
		33μg	13 ± 4^U M	14 ± 2	29 ± 4	130 ± 10	56 ± 8
		100μg	11 ± 1^U M	9 ± 4	36 ± 2	153 ± 16	63 ± 9
		333μg	14 ± 3^U M	8 ± 1	33 ± 7	139 ± 11	68 ± 9
		1000μg	14 ± 1^U M	7 ± 0	18 ± 1	141 ± 2	69 ± 2
		2500μg	10 ± 1^D M U	5 ± 1^D M	18 ± 2^D M	116 ± 4^D M	43 ± 4^D M
		5000μg	10 ± 4^D M U	5 ± 2^D M	20 ± 3^D M	102 ± 11^D M	47 ± 3^D M
	2-AA	2.5μg	383 ± 34	381 ± 75	1513 ± 175	2476 ± 79
	2-AA	10.0μg					234 ± 20

 

 

 

 

Summary of Results Experiment II

 

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	Deionised water		18 ± 6	13 ± 1	24 ± 4	130 ± 10	52 ± 7
	Untreated		19 ± 4	11 ± 4	25 ± 2	130 ± 11	48 ± 7
	FAT 40849/A TE	3μg	15 ± 5	12 ± 3	32 ± 3	122 ± 4	52 ± 7
		10μg	17 ± 4	10 ± 3	29 ± 7	125 ± 9	44 ± 3
		33μg	17 ± 1	15 ± 6	32 ± 4	129 ± 7	50 ± 11
		100μg	14 ± 2	10 ± 3	33 ± 6	136 ± 3	60 ± 6
		333μg	15 ± 3	10 ± 3	31 ± 6	132 ± 4	47 ± 7
		1000μg	11 ± 4	9 ± 1	24 ± 4	118 ± 23	38 ± 11
		2500μg	14 ± 4^D M	6 ± 1^D M	19 ± 3^D M	133 ± 8^D M	49 ± 6^D M
		5000μg	10 ± 4^D M	7 ± 4^D M	20 ± 7^D M	156 ± 10^D M	60 ± 6^D M
	NaN3	10μg	1925 ± 76			2118 ± 39
	4-NOPD	10μg			382 ± 68
	4-NOPD	50μg		113 ± 10
	MMS	3.0μL					676 ± 57
With Activation	Deionised water		18 ± 5	16 ± 9	37 ± 4	156 ± 24	67 ± 14
	Untreated		16 ± 4	17 ± 9	36 ± 7	155 ± 7	58 ± 8
	FAT 40849/A TE	3μg	21 ± 8	17 ± 4	38 ± 6	146 ± 15	57 ± 9
		10μg	18 ± 8	16 ± 1	41 ± 1	159 ± 9	56 ± 14
		33μg	18 ± 4	14 ± 2	32 ± 8	154 ± 8	64 ± 9
		100μg	22 ± 7	16 ± 5	33 ± 8	160 ± 17	59 ± 10
		333μg	14 ± 2	19 ± 5	31 ± 5	162 ± 5	78 ± 11
		1000μg	14 ± 6	11 ± 3	20 ± 2	136 ± 10	44 ± 1
		2500μg	14 ± 4^D M	8 ± 1^D M	17 ± 2^D M	134 ± 7^D M	60 ± 9^D M
		5000μg	14 ± 6^D M	6 ± 2^D M	16 ± 3^D M	150 ± 3^D M	74 ± 4^D M
	2-AA	2.5μg	346 ± 10	234 ± 28	2089 ± 162	2274 ± 50
	2-AA	10.0μg					399 ± 21

 

 

 

 

Key to Positive Controls	Key to Plate Postfix Codes
NaN3 sodium azide	D Densely coloured plate
2-AA 2-aminoanthracene	M Manual count
4-NOPD 4-nitro-o-phenylene-diamine	U Air bubbles
MMS methyl methane sulfonate

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

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, FAT 40849/A TE is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of FAT 40849/A TE to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

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

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

The plates incubated with the test item showed normal background growth up to 5000 Lg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in almost all strains the with and without metabolic activation. A minor reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 1537 and TA 98.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40849/A TE at any dose 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 and showed a distinct increase of induced revertant colonies.