Disodium {sulfonylbis[4,1-phenylenediazene-2,1-diyl(1-ethyl-6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-5,3-diyl)]}dimethanesulfonate

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:

2010 -09-20 till 2010-10-22

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

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:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Details on test material:

- Name of test material (as cited in study report): Acid Yellow RN 2903

- Physical state: Solid
- Purity test date: Approx. 65 %
- Lot/batch No.: Vers. Kilo 6,26.05.2009
- Expiration date of the lot/batch: 2015-08-01
- Stability under test conditions: 4 hours in water, polyethylene glycol, CMC at room temperature
- Storage condition of test material: Room temperature
- Other:

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 / non induced hamster liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar, plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- 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

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 in the test tubes at 2500 and 5000 µg/plate and on the incubated agar plates from 1000 - 5000 µg/plates. The undissolved particles had no influence on the data recording.
- Other confounding effects:

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation with the exception of strain TA 98 with S9 mix where a minor toxic effect was observed at 5000 µg/plate in experiment II.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tabellen

Pre-Experiment/Experiment I

Study Name: 1368902	Study Code: Harlan CCR 1368902
Experiment: 1368902 VV Plate	Date Plated: 20/09/2010
Assay Conditions:	Date Counted: 23/09/2010

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 ± 3	10 ± 1	39 ± 3	189 ± 9	50 ± 4
	Untreated			11 ± 2	13 ± 0	39 ± 7	198 ± 4	61 ± 2
	Acid Yellow RN	3 µg		13 ± 1	9 ± 3	44 ± 8	181 ± 13	50 ± 2
	2903	10 µg		12 ± 2	13 ± 2	38 ± 10	193 ± 7	57 ± 1
		33 µg		12 ± 2	10 ± 3	30 ± 3	206 ± 8	54 ± 9
		100 µg		18 ± 4	13 ± 1	40 ± 5	202 ± 5	50 ± 12
		333 µg		13 ± 7^P	11 ± 3^P	34 ± 2^P	201 ± 10^P	51 ± 4^P
		1000 µg		15 ± 4^P	11 ± 4^P	33 ± 1^P	198 ± 9^P	52 ± 5^P
		2500 µg		13 ± 4^P	9 ± 1^P	33 ± 5^P	200 ± 10^P	54 ± 4^P
		5000 µg		10 ± 4^P M	6 ± 3^P	26 ± 2^P	133 ± 12^P	43 ± 7^P
	NaN3	10 µg		1661 ± 22			1802 ± 57
	4-NOPD	10 µg				342 ± 14
	4-NOPD	50 µg			99 ± 12
	MMS	3.0 µL						1350 ± 21
With Activation	Deionised water			14 ± 1	12 ± 0	42 ± 6	185 ± 25	66 ± 19
	Untreated			18 ± 9	15 ± 4	50 ± 15	175 ± 9	62 ± 5
	Acid Yellow RN	3 µg		15 ± 4	16 ± 2	41 ± 7	186 ± 19	64 ± 9
	2903	10 µg		18 ± 2	12 ± 3	46 ± 7	190 ± 5	51 ± 3
		33 µg		16 ± 4	14 ± 1	37 ± 2	201 ± 18	63 ± 11
		100 µg		23 ± 7	10 ± 3	37 ± 3	194 ± 2	59 ± 10
		333 µg		16 ± 4^P	9 ± 3^P	37 ± 5^P	190 ± 6^P	62 ± 5^P
		1000 µg		20 ± 4^P	14 ± 6^P	35 ± 8^P	212 ± 13^P	58 ± 6^P
		2500 µg		17 ± 1^P	11 ± 4^P	41 ± 3^P	216 ± 11^P	64 ± 4^P
		5000 µg		17 ± 4^P M	7 ± 1^P M	19 ± 2^P M	153 ± 11^P M	45 ± 7^P M
	2-AA	2.5 µg		433 ± 25	309 ± 22	2471 ± 212	2673 ± 31
	2-AA	10.0 µg						283 ± 19

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

Experiment II

Study Name: 1368902	Study Code: Harlan CCR 1368902
Experiment: 1368902 HV2 Pre	Date Plated: 05/10/2010 / 19/10/2010*
Assay Conditions:	Date Counted: 08/10/2010 / 22/10/2010*

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			15 ± 2	9 ± 4	20 ± 2	177 ± 7	69 ± 6
	Untreated			14 ± 2	7 ± 3	24 ± 2	200 ± 12	68 ± 7
	Acid Yellow RN	3 µg		14 ± 2	10 ± 2	26 ± 3	185 ± 15	71 ± 14
	2903	10 µg		16 ± 3	11 ± 7	27 ± 7	183 ± 6	62 ± 7
		33 µg		13 ± 3	11 ± 3	17 ± 2	185 ± 13	85 ± 11
		100 µg		22 ± 3	8 ± 2	20 ± 3	187 ± 6	76 ± 13
		333 µg		14 ± 2	10 ± 1	26 ± 4	192 ± 8	62 ± 7
		1000 µg		16 ± 3^P	9 ± 1^P	28 ± 7^P	193 ± 8^P	65 ± 7^P
		2500 µg		17 ± 5^P	9 ± 4^P	25 ± 4^P	185 ± 7^P	72 ± 1^P
		5000 µg		13 ± 1^P	7 ± 2^P	25 ± 4^P	160 ± 17^P	74 ± 10^P
	NaN3	10 µg		1743 ± 48			1598 ± 57
	4-NOPD	10 µg				529 ± 115
	4-NOPD	50 µg			73 ± 21
	MMS	3 µL						1048 ± 16
With Activation	Deionised water			11 ± 2	7 ± 2	31 ± 9	157 ± 3	52 ± 7
	Untreated			17 ± 4	6 ± 2	37 ± 6	143 ± 14	57 ± 5
	Acid Yellow RN	3 µg		11 ± 2	7 ± 1	40 ± 6	152 ± 9	55 ± 9
	2903	10 µg		10 ± 8	7 ± 3	34 ± 4	166 ± 12	57 ± 8
		33 µg		12 ± 4	8 ± 2	26 ± 3	155 ± 10	50 ± 15
		100 µg		12 ± 3	9 ± 4	31 ± 4	167 ± 11	60 ± 6
		333 µg		17 ± 2	6 ± 1	45 ± 5	172 ± 14	63 ± 8
		1000 µg		15 ± 4^P	6 ± 2^P	40 ± 1^P	231 ± 6^P	70 ± 7^P
		2500 µg		14 ± 5^P	11 ± 4^P	23 ± 4^P M	203 ± 17^P	64 ± 8^P
		5000 µg		12 ± 2^P M	6 ± 2^P M	13 ± 2^P M	135 ± 20^P M	53 ± 7^P M
	2-AA	2.5 µg		213 ± 28	151 ± 29		3189 ± 55
	2-AA	2.5 µg
	2-AA	10 µg						715 ± 73
	Congo red	500 µg				249 ± 2

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 N	Precipitate Manual count Analysis not possible

* = repeated experiment

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutage¬nicity 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.

Executive summary:

This study was performed to investigate the potential of Acid Yellow RN 2903 to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100 and theEscherichia colistrain WP2uvrA.

The assay was performed in two independent experiments with and without liver microsomal activation. Due to contamination, analysis of strain TA 98 was not possible in experiment II. This part of the experiment was repeated under identical conditions. The repeated part is reported as experiment II. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

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

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation with the exception of strain TA 98 with S9 mix where a minor toxic effect was observed at 5000 µg/plate in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Acid Yellow RN 2903 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. They showed a distinct in­crease of induced revertant colonies.

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.