2,5-dichloro-4-[[2-(dibutylamino)-4-methyl-6-[[2-(4-sulphophenyl)ethyl]amino]-5-pyrimidinyl]azo]benzenesulphonic acid, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item has no mutagenic activity on the applied bacterium tester strains, under the tested conditions.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 22 to March 03, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Principles of method if other than guideline:

The test item belongs to azo-dyes therefore in the Confirmatory Mutation Test (Pre-Incubation Test) a modified protocol (using uninduced hamster liver S9, modified co-factor and S9 mixture, longer pre-incubation) proposed by Prival and Mitchell was applied

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Supplier: Trinova Biochem GmbH
- Periodical check: the phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al.
- Spontaneous reversions: spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
- Procedure for Bacterial Cultures: the frozen bacterial cultures were thawed at room temperature and 200 µl inoculum was used to inoculate each 50 ml of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 11-14 hours in a 37 °C Benchtop Incubator Shaker.
- Viability and the Cell Count of the Testing Bacterial Cultures: fresh cultures of bacteria were grown up to the late exponential or early stationary phase of growth (approximately 10^9 cells per ml). Cultures in late stationary phase were not used. The viability of each testing culture was determined by plating 0.1 ml of the 10^-5, 10^-6, 10^-7 and 10^-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Metabolic activation:

with and without

Metabolic activation system:

rat and hamster liver S9

Test concentrations with justification for top dose:

Initial and Confirmatory Mutation Tests: 5000; 1600; 500; 160; 50, 16 and 5 µg/plate.

Vehicle / solvent:

TEST ITEM
- Vehicle: ultrapure water.
- Justification for choice of vehicle: the behaviour of the test item as a solute in top agar and in phosphate buffer was determined in the preliminary Solubility Test and ultrapure water was found as suitable vehicle for preparing the test item solutions. In the preliminary Concentration Range Finding Test ultrapure water was used for preparing the test item solutions and short (<30 min) slight heating (up to 37 °C) facilitated the test item dissolution.

CONTROL
- Solvents: DMSO was applied as vehicle for positive control substances 4-Nitro-1,2-phenylenediamine, 9-Aminoacridine and 2-aminoanthracene.

Untreated negative controls:

yes

Remarks:

ultrapure water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

congo red

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine; 2-aminoanthracene

Details on test system and experimental conditions:

The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).
In the preliminary Concentration Range Finding Test as well as in the Initial Mutation Test the plate incorporation method was used.
The Confirmatory Mutation Test was performed using the modified test design developed by Prival and Mitchell.

INITIAL MUTATION TEST
- Method: standard plate incorporation. Molten top agar was prepared and kept at 45 °C. 2 ml of top agar was aliquoted into individual test tubes. The test item and other components were prepared fresh and added to the overlay (45°C).
- Replicates: 3 tubes per both control and each concentration level.
- Culture: in Nutrient Broth No.2., bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
- Composition: the typical content of the tubes: top agar 2000 µl, vehicle or solution of test item or positive controls 100 µl, overnight culture of test strain (containing approximately 10^8 viable cells) 100 µl, phosphate buffer (pH: 7.4) or S9 mix 500 µl. For activation studies, instead of phosphate buffer, 0.5 ml of the S9 Mix (containing rat liver S9) was added to each overlay tube.
- Incubation conditions: the plates were incubated at 37 °C for about 48 hours.

CONFIRMATORY MUTATION TEST
- Method: pre-incubation procedure.
- Replicates: triplicate plating was used at each dose level.
- Culture: before the overlaying of the test item, the bacterial culture (0.1 ml, containing approximately 10^8 viable cells) and the S9 mix (containing uninduced hamster liver S9) or phosphate buffer (0.5 ml) was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle).
- Pre-incubation conditions: tubes were gently mixed and incubated for 30 min at 37 ºC in a shaking incubator.
- Composition: after the incubation period, 2 ml of molten top agar was added to the tubes, the content mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method. Tubes were aerated during pre-incubation by using a shaker.
- Incubation conditions: after preparation the plates were incubated at 37 °C for about 48 hours.

SOLUBILITY TEST
- Preparations of test solution: the test item was completely dissolved (and diluted accordingly) in ultrapure water (for facilitating the test item dissolution a short slight heating was applied).
- Test system: the obtained solutions with the solution of top agar and phosphate buffer were examined in a test tube without test bacterium suspension.
- Concentration of test item: 5000 µg/tube

CONCENTRATION RANGE FINDING TEST (Informatory Toxicity Test)
- Dilution factor: the stock solution with a concentration of 50 mg/ml was prepared in ultrapure water and diluted in at least 6 steps by factor of approximately √10.
- Test concentrations: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.

MEDIA USED
The Minimal Glucose Agar (MGA) Plates
- Supplier: VWR International
- Typical composition (g/1000 ml) of MGA plates: Glucose 20.0 g, Magnesium sulfate 0.2 g, Citric acid 2.0 g, di-Potassium hydrogenphosphate 10.0 g, Sodium ammonium hydrogenphosphate 3.5 g, Agar agar 13.0 g

Nutrient broth No. 2
- Composition: Nutrient broth No. 2. 25.0 g, Ultrapure water ad 1000.0 ml.
- Preparation: sterilization for 20 minutes was performed at 121 ˚C in an autoclave.

Nutrient Agar
- Composition: Nutrient Agar 20.0 g, Ultrapure water ad 1000.0 ml.
- Preparation: sterilization for 20 minutes was performed at 121˚C in an autoclave.

Top Agar for Salmonella typhimurium Strains
- Agar solution: Agar Bacteriological 4.0 g, NaCl 5.0 g, Ultrapure water ad 1000.0 ml.
- Preparation: sterilization for 20 minutes was performed at 121˚C in an autoclave.
- Histidine – Biotin solution (0.5 mM): D-Biotin 122.2 mg, L-Histidine x HCl H2O 104.8 mg , Ultrapure water ad 1000.0 ml.
- Preparation: sterilization was performed by filtration through a 0.22 µm membrane filter.
- Complete Top Agar for Salmonella typhimurium strains: Histidine – Biotin solution (0.5 mM) 100.0 ml and Agar solution 900.0 ml.

Top Agar for Escherichia coli Strain
- Tryptophan solution (2 mg/ml): L-Tryptophan 2000.0 mg, Ultrapure water ad 1000.0 ml.
- Preparation: sterilization was performed by filtration through a 0.22 µm membrane filter.
- Complete Top Agar for Escherichia coli strain: Nutrient Broth by 5.4.2 50.0 ml, Tryptophan solution (2 mg/ml) 2.5 ml, Agar solution by 5.4.4 947.5 ml.

RAT LIVER FRACTION
- The S9 Mix (with Rat Liver S9): NADP Na 7.66 g (4 mM), D-glucose-6 phosphate Na 3.53 g (5 mM), MgCl2 1.90 g (8 mM), KCl 6.15 g (33 mM), Ultrapure water ad 1000 ml.
- Preparation: sterilized by filtration through a 0.22 µm membrane filter.
- The complete S9 Mix was freshly prepared containing components as follows: Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 ml, Rat liver homogenate (S9) 100 ml, Salt solution for S9 Mix 400 ml.
- Storage: the S9 Mix was kept in an ice bath before it was added to the culture medium.

HAMSTER LIVER FRACTION
- The S9 Mix (with hamster Liver S9): NADP Na 15.31 g (4 mM), NADH Na2 x H2O 7.27 g (2 mM), FMN (Riboflavine-5’-phosphate-sodium salt) x H2O 4.96 g (2 mM), D-glucose-6 phosphate Na 28.20 g (20 mM), MgCl2 3.80 g (8 mM), KCl 12.31 g (33 mM), Ultrapure water ad 1000 ml.
- Preparation: sterilized by filtration through a 0.22 µm membrane filter.
- The complete S9 Mix was freshly prepared containing components as follows (per 1000 ml): Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 ml, Hamster liver homogenate (S9) 300 ml, Salt solution for S9 Mix 200 ml, D-glucose-6-phosphate-dehydrogenase 2800 U.
- Storage: before adding to the culture medium the S9 Mix was kept in an ice bath.

VALIDITY OF THE TEST
The tests (Initial and Confirmatory Mutation Tests) are considered to be valid if:
- all of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- the Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- the Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- the bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- the tester strain culture titers is in the 109 cells/mL order.
- the batch of S9 used in this study shows the appropriate biological activity.
- the reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- there are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

A dose level is considered toxic if:
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.

Rationale for test conditions:

Choice of the concentrations was done on the basis of a Solubility Test and a Concentration Range Finding Test (Informatory Toxicity Test).

Evaluation criteria:

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

ultrapure water

Untreated negative controls validity:

valid

Remarks:

ultrapure water

Positive controls validity:

valid

Additional information on results:

INITIAL AND CONFIRMATORY MUTATION TESTS (PLATE INCORPORATION AND PRE-INCUBATION TESTS)
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in S. typhimurium TA1537 strain, at 5000 μg/plate, in the absence of metabolic activation (S9). This value however remained in the range of the corresponding vehicle historical control data and additional concentration related increase in revertant colony counts was not noticed. The mutation rate was 1.67, which was far below the genotoxicological threshold for being positive.
In the Initial and Confirmatory Mutation Tests, an unequivocal inhibitory effect of the test item on bacterial growth was observed. Decreased revertant colony counts, revertants below the vehicle and corresponding historical control data ranges indicated the cytotoxicity in the S. typhimurium TA98 and TA100 strains. The affected background lawn development noticed in the Concentration Range Finding Test was not confirmed, repeated in the subsequent main experiments.

In the Initial and Confirmatory Mutation Tests cytotoxicity (with above listed signs) was noticed in S. typhimurium TA98 at the concentrations of 5000 and 1600 µg/plate and in TA100 in the concentration range of 5000-500 µg/plate, in the absence of exogenous metabolic activation ( S9 Mix); furthermore in the Initial Mutation Test in both strains, in the Confirmatory Mutation Test in S. typhimurium TA98 at 5000 µg/plate in the presence of exogenous metabolic activation (+S9 Mix) .
No precipitation of the test item was observed in the Initial and Confirmatory Mutation Tests on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).

CONCENTRATION RANGE FINDING TEST (Informatory Toxicity Test)
The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
In the Informatory Toxicity Test inhibitory effect of the test item was observed, indicated by affected colony and background lawn development. Reduced revertant growth and additionally slightly reduced background lawn development was noticed in both strains at the highest examined concentrations of 5000 and 1600 µg/plate in absence ( S9 Mix) and at 5000 µg/plate in the presence of exogenous metabolic activation (+S9 Mix). Further indication of the unequivocal inhibition that the obtained revertant colony numbers were below the vehicle and corresponding historical control data range, but the background lawn development was not affected in S. typhimurium TA100 at 500 µg/plate ( S9 Mix).
The revertant colony numbers were below the revertant colony numbers of the vehicle control; however remained within the historical control data range in S. typhimurium TA98 at 500 µg/plate, in absence of exogenous metabolic activation ( S9 Mix).
Slightly higher revertant colony counts within the corresponding historical control data ranges were obtained in S. typhimurium TA98 at 16 and 5 µg/plate in presence of exogenous metabolic activation (+S9 Mix); furthermore in TA100 at 50 µg/plate (+S9 Mix).
No precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9 Mix).

CONTROLS
In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the ultrapure water vehicle control plates with and without S9 Mix were in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. In the Initial Mutation Test, in the case of S. typhimurium TA100 the revertant colony numbers of 2-Aminoanthracene (2AA); furthermore in the Confirmatory Mutation Test in the case of E. coli WP2 uvrA the revertant colony numbers of Methyl methanesulfonate (MMS), in the case of S. typhimurium TA1537 the revertant colony numbers of the 2-Aminoanthracene (2AA) were above the corresponding historical control data ranges; however these were considered as acceptable without any effect on the final conclusion of the study.

The revertant colony numbers of the untreated and dimethyl sulfoxide (DMSO) control plates in different experimental phases were slightly higher or lower than the ultrapure water control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.
In summary, the actual values of untreated, vehicle and positive controls were in line with the criteria for validity of the assay.

VALIDITY OF THE EXPERIMENT
The tester strains used demonstrated the specific phenotype characteristics, were in line with the corresponding historical control data ranges and showed the adequate strain culture titer. Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system.
Each of the investigated reference mutagens showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in both main experimental phases and the number of revertants in most cases fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in the main experimental phases, in all tester strains.
The spontaneous revertant colony numbers of the ultrapure water vehicle control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in both main experimental phases.
Seven concentration levels were investigated in the Informatory Toxicity Test and in the main mutation experiments (Initial and Confirmatory Mutation Tests).
In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain.
All criteria for the validity of the performed experiments have therefore been met.

Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichiacoli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	25	1.15	32.3	1.08	117	1.2	141	1.23	10.7	0.82	14.7	1.47	9	0.9	10	1.03	26.7	1.04	33.7	0.98
DMSO Control	25	1	22.3	1	–	–	124.7	1	–	–	11	1	10.7	1	15.3	1	–	–	33.3	1
Ultrapure Water Control	21.7	1	30	1	97.7	1	115	1	13	1	10	1	10	1	9.7	1	25.7	1	34.3	1
5000	9.3	0.43	5.3	0.18	3.3	0.03	12	0.1	6	0.46	7.3	0.73	5.3	0.53	4.7	0.48	17.3	0.68	30.3	0.88
1600	5.7	0.26	21.7	0.72	5.3	0.05	102	0.89	12	0.92	11.3	1.13	10.3	1.03	8	0.83	23.7	0.92	27.3	0.8
500	20.3	0.94	25	0.83	58.7	0.6	101.3	0.88	12.7	0.97	11	1.1	9	0.9	7.3	0.76	18	0.7	36.7	1.07
160	25	1.15	28.7	0.96	108	1.11	101	0.88	12.3	0.95	12.7	1.27	10.3	1.03	8	0.83	20.7	0.81	31.7	0.92
50	20.7	0.95	25.3	0.84	102.7	1.05	109.7	0.95	13	1	12	1.2	8.3	0.83	10.3	1.07	22.7	0.88	36.3	1.06
16	28	1.29	30	1	106.7	1.09	112.3	0.98	13.7	1.05	10.7	1.07	9	0.9	9	0.93	22.3	0.87	33.3	0.97
5	28	1.29	29	0.97	120	1.23	120	1.04	14	1.08	12.3	1.23	10.3	1.03	13.7	1.41	22.3	0.87	35	1.02
NPD (4 mg)	184	7.36	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 mg)	–	–	–	–	1077.3	11.03	–	–	890.7	68.51	–	–	–	–	–	–	–	–	–	–
9AA (50 mg)	–	–	–	–	–	–	–	–	–	–	–	–	944	88.5	–	–	–	–	–	–
MMS (2 ml)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	941.3	36.68	–	–
2AA (2 mg)	–	–	1616	72.36	–	–	3034.7	24.34	–	–	190.7	17.33	–	–	172	11.22	–	–	–	–
2AA (50 mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	190.7	5.72

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances SAZ and MMS; the DMSO was applied as vehicle for positive control substances NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimurium tester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	17.7	0.66	34.3	0.84	109	1.15	104.3	0.83	12	1	12	1	4.7	0.93	10.3	1.11	17	0.69	24.7	0.95
DMSO Control	20	1	34	1	–	–	88.3	1	–	–	12.3	1	6.3	1	8	1	–	–	22.7	1
Ultrapure Water Control	26.7	1	41	1	95	1	126	1	12	1	12	1	5	1	9.3	1	24.7	1	26	1
5000	4.7	0.18	36.3	0.89	5.3	0.06	122	0.97	12	1	13.3	1.11	8.3	1.67	8.3	0.89	32.7	1.32	15	0.58
1600	4.3	0.16	34	0.83	2.3	0.02	124	0.98	12.3	1.03	13.7	1.14	5.3	1.07	9.7	1.04	31.3	1.27	20.3	0.78
500	14	0.53	38.3	0.93	50.7	0.53	129.3	1.03	12.3	1.03	10.7	0.89	4.7	0.93	8	0.86	34.3	1.39	22	0.85
160	23.7	0.89	41.3	1.01	92.7	0.98	115.3	0.92	12.3	1.03	10	0.83	5	1	7	0.75	27.3	1.11	22	0.85
50	27	1.01	44	1.07	106.3	1.12	107.3	0.85	14.7	1.22	11.7	0.97	7.3	1.47	10	1.07	27.3	1.11	22.3	0.86
16	22.3	0.84	33.3	0.81	100.3	1.06	114.7	0.91	13	1.08	12.3	1.03	6.3	1.27	12.3	1.32	29.7	1.2	26.3	1.01
5	22.7	0.85	38.3	0.93	103.3	1.09	117.3	0.93	10.7	0.89	10.3	0.86	5	1	11.3	1.21	25.3	1.03	22	0.85
NPD (4 mg)	226.3	11.32	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 mg)	–	–	–	–	933.3	9.82	–	–	925.3	77.11	–	–	–	–	–	–	–	–	–	–
9AA (50 mg)	–	–	–	–	–	–	–	–	–	–	–	–	508	80.21	–	–	–	–	–	–
MMS (2 ml)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1544	62.59	–	–
Congo Red (250 mg)	–	–	340	10	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
2AA (2 mg)	–	–	–	–	–	–	2794.7	31.64	–	–	428.7	34.76	–	–	407.3	50.92	–	–	–	–
2AA (50 mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	350	15.44

MR: Mutation Rate;  NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances Congo Red, SAZ and MMS; the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, Congo Red, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Summary Table of the Results of the Concentration Range Finding Test

Range Finding Test (Informatory Toxicity Test)
Concentrations (mg/plate)	Salmonella typhimurium tester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Mutation rate (MR)
Untreated Control	18	0.75	28.3	1.06	100.7	1.11	112	0.95
DMSO Control	17.3	1	28	1	–	–	101	1
Ultrapure Water Control	24	1	26.7	1	90.3	1	118	1
5000	3.3	0.14	8	0.3	5.7	0.06	24.7	0.21
1600	7.7	0.32	22.7	0.85	4.3	0.05	103	0.87
500	13	0.54	27.7	1.04	57.3	0.63	107	0.91
160	24.7	1.03	27.3	1.03	88	0.97	111.7	0.95
50	26	1.08	31.3	1.18	109.3	1.21	111.3	0.94
16	25	1.04	34.7	1.3	108	1.2	135.3	1.15
5	25	1.04	35.3	1.33	107	1.18	123.3	1.05
NPD (4 mg)	280	16.15	–	–	–	–	–	–
SAZ (2 mg)	–	–	–	–	1130.7	12.52	–	–
2AA (2 mg)	–	–	1586.7	56.67	–	–	2349.3	23.26

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 2AA: 2-aminoanthracene

Conclusions:

The test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item was asssessed with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The test item belongs to azo-dyes therefore in the Confirmatory Mutation Test (Pre-Incubation Test) a modified protocol (using uninduced hamster liver S9, modified co-factor and S9 mixture, longer pre-incubation) proposed by Prival and Mitchell was applied.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (strains TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9). In the plate incorporation test (Initial Mutation Test) S9 prepared from livers of phenobarbital/β-naphthoflavone-induced rats was used, in the pre-incubation test S9 prepared from uninduced hamster liver was used.

The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests: 5000; 1600; 500; 160; 50, 16 and 5 µg/plate.

The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and nearly all the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

In the performed experiments unequivocal inhibitory effect of the test item was observed. In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity (noticed following plate incorporation and pre-incubation procedures in Salmonella typhimurium TA100 strain in the absence of metabolic activation).

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.

Conlcusion

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN VITRO GENE MUTATION IN BACTERIA

The test item was asssessed with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The test item belongs to azo-dyes therefore in the Confirmatory Mutation Test (Pre-Incubation Test) a modified protocol (using uninduced hamster liver S9, modified co-factor and S9 mixture, longer pre-incubation) proposed by Prival and Mitchell was applied. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (strains TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9). In the plate incorporation test (Initial Mutation Test) S9 prepared from livers of phenobarbital/β-naphthoflavone-induced rats was used, in the pre-incubation test S9 prepared from uninduced hamster liver was used. Based on the results of the preliminary range finding test the following concentrations of the test item were prepared and investigated: 5000; 1600; 500; 160; 50, 16 and 5 µg/plate. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (± S9 Mix) throughout the study.

In the performed experiments unequivocal inhibitory effect of the test item was observed. In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity (noticed following plate incorporation and pre-incubation procedures in Salmonella typhimurium TA100 strain in the absence of metabolic activation). No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA, under the tested conditions

Justification for classification or non-classification

According to the CLP Regulation (EC ) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC) No 1272/2008.