Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(3-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: non mutagenic

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 August 03 to September 29, 2018

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 on 21st July 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Molecular Toxicology, Inc. PO Box 1189 Boone, NC 28607 USA.
- Stock storage: stock cultures in oxoid nutrient broth no. 2 were stored in the test facility as frozen permanents in -80 ± 10 ºC. Laboratory stocks of each strain were maintained on minimal glucose agar as master plates. These master plates were stored in a refrigerator between 2 to 8 ºC for 3 months

GENETIC CHARACTERIZATION OF TEST STRAINS
After preparation of the master plates, the growth requirements and the genetic identity of strains like histidine requirement, sensitivity to UV radiation and resistance of strains TA98, TA100, TA1535, TA1537 and TA102 to ampicillin, resistance of TA102 for tetracycline and rfa mutation of Salmonella typhimurium strains were checked along with the range of spontaneous revertants.

Metabolic activation:

with and without

Metabolic activation system:

S9 rat and hamster liver fraction

Test concentrations with justification for top dose:

MUTAGENICITY ASSAY: 0.01, 0.03, 0.10, 0.32 and 1 mg a.i/plate were selected (with half-log dose interval), both experiment I and II, with and without metabolic activation
CYTOTOXICITY: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg a.i/plate

Vehicle / solvent:

- Vehicle: distilled water.
- Justification for choice of /vehicle: the solubility test was carried out with distilled water, dimethyl sulphoxide, ethanol and acetone. A quantity of 500 mg of test item was mixed with 10 ml of respective solvents and vortexed. The test item was found to be non soluble in dimethyl sulphoxide, ethanol and acetone. On the contrary, test item formed suspension with distilled water at 50 mg a.i/ml (1.070 g of test item in 10 ml was taken considering purity correction factor).

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
Experiment I - plate incorporation method.
- In presence of metabolic activation: 2.0 ml soft agar containing histidine-biotin, 500 µl S9 mix, 100 µl test concentrations /vehicle/appropriate positive control, 100 µl bacterial culture (containing approximately 10^8 viable cells).
- In absence of metabolic activation: 2.0 ml soft agar containing histidine-biotin, 500 µl Phosphate Buffer Saline (PBS), 100 µl test concentrations /vehicle/appropriate positive control, 100 µl bacterial culture (containing approximately 10^8 viable cells).
- Incubation: after soft agar set, the plates were incubated at 37±1 ºC for 65 hours and 40 minutes.
- Count: the revertant colonies were counted manually and the plates were examined for bacterial background lawn.

Experiment II - preincubation method.
- In presence of metabolic activation: 500 µl S9 mix, 100 µl test concentrations/vehicle/ appropriate positive control, 100 µl bacterial culture (containing approximately 10^8 viable cells).
- In absence of metabolic activation: 500 µl Phosphate Buffer Saline (PBS), 100 µl test concentrations, /vehicle/appropriate positive control, 100 µl bacterial culture (containing approximately 10^8 viable cells).
- Incubation: preparations were kept in an incubator shaker at 180 rpm for 20 minutes at 37±1 ºC. After the incubation period, 2 ml of molten soft agar containing histidine-biotin for Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 tester strains was added to each of the tubes. The tube contents were mixed and overlaid onto minimal glucose agar plates. After the soft agar sets, the plates were incubated at 37±1 ºC for 65 hours and 12 minutes.
- Count: the revertant colonies were counted manually and the plates were examined for bacterial background lawn.

NUMBER OF REPLICATIONS: triplicate.

VIABLE COUNT
The bacterial suspension of each tester strain was diluted up to 10^-7 in phosphate buffer saline and 1000 µl of the diluted suspension from each tester strain was plated onto nutrient agar plates in triplicate. The plates were incubated at 37±1 ºC for 24 hours and 15 minutes for plate incorporation method for pre incubation method, respectively. After incubation, the number of colonies in each plate were counted manually and expressed as number of Colony Forming Units per ml (CFU/ml) of the bacterial suspension.

COLONY COUNT OF REVERTANT
Revertant colonies for a respective strain, within the test item dilution series were counted manually.

QUALITY CONTROL PLATES
Control plates with S9 mix, PBS, test item, soft agar, distilled water, dimethyl sulphoxide and minimal glucose agar plates were evaluated for contamination.

PRECIPITATION TEST
Stock solution of test item was serially diluted to get the different concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 and 50 mg a.i/ml using distilled water. A quantity of 100 µl of different concentrations of test item was separately mixed with 2 ml of molten soft agar, vortexed and spread onto minimal glucose agar plates to achieve the concentrations 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg a.i/plate. Plates were incubated for 2 hours at 37±1 °C.

CYTOTOXICITY
Based on the results of precipitation test, an initial cytotoxicity test was conducted for the selection of test concentrations for the mutation assay. Salmonella typhimurium TA100 tester strain was exposed to concentrations 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg a.i/plate of test item in triplicate, both in the presence and absence of reductive metabolic activation along with concurrent vehicle control (distilled water).
Each concentration of test item was mixed with soft agar containing histidine and biotin, S9 mix (for presence of reductive metabolic activation), phosphate buffer saline (for absence of reductive metabolic activation), Salmonella typhimurium TA100 of cell density approximately 18 × 10^8 cells/ml and overlaid on to prelabeled minimal glucose agar plates. The plates were incubated at 37±1 ºC for 71 hours.

PREPARATION OF S9 HOMOGENATE AND ACTIVATION MIXTURE
Rat liver S9 homogenate
Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar rats induced with intraperitoneal injection of sodium phenobarbitone and β naphthoflavone at 16 and 20 mg/ml, respectively, for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10 ºC until use. Each batch of S9 homogenate was assessed for sterility by streaking the supernatant fluid on nutrient agar plates and incubated at 37±1 ºC for 48 hours. It was found sterile and was further evaluated for its protein content and for its ability to metabolize the promutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain. The results were found to be acceptable for the tested parameters.
A volume of 1 ml of S9 homogenate was thawed immediately before use and mixed with 9 ml of co-factor solution containing 4 mM Nicotinamide Adenine Dinucleotide Phosphate (NADP) disodium salt, 5 mM glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in phosphate buffer saline (PBS) of and pH 7.33 for plate incorporation to get the concentration of 10 % (v/v).

Hamster liver S9 homogenate
The S9 homogenate of hamster liver was procured from Molecular Toxicology.
For 75.5 ml S9 homogenate, flavin mononucleotide (FMN) 72.48 mg in 5.3 ml of water (filter sterilized and kept on ice pack) to a 250 ml conical flask, co-factors were added that have been allowed to thaw at room temperature. 319.4 mg of D-glucose-6-phosphate and 107.2 mg of nicotine adenine dinucleotide (NADH) were added they were once dissolved filter sterilization was done through 0.45 µm filter. Aseptically 1 mg of glucose-6-phosphate dehydrogenase was added to the co-factor mixture in phosphate buffer saline (PBS) pH 7.32 for initial cytotoxicity and 7.33 for preincubation method followed by 22.7 ml of liver S9 fraction. Just prior to performing the experiment the FMN solution was added. The final mixture was kept on ice for the duration of experiment. The post-mitochondrial fraction used at concentration was in the range of 30 % v/v in the S9-mix.

CRITERIA FOR ACCEPTABILITY OF THE TEST
The mutation test is considered acceptable as it meets the following criteria:
- all tester strains confirmed to their genetic characteristics.
- the positive controls showed increase in revertant colony numbers of at least twice or thrice the concurrent vehicle control levels with the appropriate bacterial strain.

Evaluation criteria:

The conditions necessary for determining a positive result are: there should be a dose related increase over the range tested and/or a reproducible increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item, either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited.
A response is evaluated as negative, as it is neither positive nor equivocal.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Plate Incorporation Method - experiment I
All the tester strains treated with test item showed very close resemblance to the vehicle control when tested with and without metabolic activation. There was no appreciable increase in number of revertant colonies and no change in bacterial background lawn when compared to that of the vehicle control, among the tester strains. The mean number of revertant colonies/plate and bacterial background lawn in the treatment groups for the tested strains were comparable to that of vehicle control.
The specific positive controls tested simultaneously produced approximately 2.2 to 14.7 fold increase in mean number of revertants as compared to the vehicle control.

Preincubation Method - experiment II
All the tester strains treated with test item showed very close resemblance to the vehicle control when tested with and without metabolic activation. There showed no appreciable increase in number of revertant colonies and no change in bacterial background lawn when compared to that of the vehicle control, among the tester strains. The mean number of revertant colonies/plate and bacterial background lawn in the treatment groups for the tested strains were comparable to that of vehicle control.
The specific positive controls tested simultaneously produced approximately 2.3 to 12.5 fold increase in mean number of revertants as compared to the vehicle control.

VIABLE COUNT
Each tester strain was serially diluted to 10^-7 and plated on nutrient agar. Post incubation for plate incorporation method and for pre incubation method, the numbers of colonies were counted manually and results were expressed as Colony Forming Units (CFU). Each tester strains resulted in acceptable range of 1 to 2×10^9 CFU/ml.

QUALITY COUNT PLATES
Control plates with S9 mix, phosphate buffer saline, soft agar, 1 mg a.i/plate of test item, distilled water, dimethyl sulphoxide and minimal glucose agar plates incubated at 37±1 °C for 65 hours and 40 minutes for plate incorporation method and for pre incubation method were checked for contamination. No microbial contamination was observed.

SOLUBILITY TEST
The test item formed suspension in distilled water at a concentration of 50 mg a.i/ml.

PRECIPITATION TEST
The test item resulted in heavy precipitation at 3, 4 and 5 mg a.i/plate, moderate precipitation at 2 mg a.i/plate, mild precipitation at 0.9 and 1 mg a.i/plate, minimal precipitation at 0.7 and 0.8 mg a.i/plate, slight precipitation at 0.6 mg a.i/plate and no precipitation at 0.4 and 0.5 mg a.i/plate was observed at the tested concentration.

INITIAL CYTOTOXICITY TEST
Based on the results of the solubility and precipitation tests, the concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg a.i/plate were selected for the initial cytotoxicity test.
On the basis of cytotoxicity results 1 mg a.i/plate was considered as the highest test concentration for mutation assay.

SUMMARY OF COLONY COUNTS OF REVERTANTS OF EXPERIMENT I - Plate Incorporation Method

Test Concentration  (mg a.i/plate)		No. of revertants (mean of 3 plates)
		with S9					without S9
		Salmonella typhimurium					Salmonella typhimurium
		TA 98	TA 100	TA 102	TA 1535	TA 1537	TA 98	TA 100	TA 102	TA 1535	TA 1537
100 µl of distilled water	Mean	25.3	107.7	266.3	20.7	9.3	24.3	97.7	261.3	18.3	8.0
	±SD	1.5	9.1	7.4	2.5	2.1	3.2	6.5	9.5	2.1	1.0
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
Test substance 0.01	Mean	24.7	103	264.3	20.0	7.7	22.0	96.3	258.3	18.7	6.0
	±SD	2.1	4.6	3.5	2.6	1.5	2.0	1.5	5.7	2.1	1.0
Fold Increase		1.0	1.0	1.0	1.0	0.8	0.9	1.0	1.0	1.0	0.8
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
Test substance 0.03	Mean	22.0	102.7	263.0	19.0	8.3	21.7	96.7	258.0	16.3	7.0
	±SD	1.0	3.2	8.5	2.0	0.6	1.2	3.2	5.6	1.5	1.0
Fold Increase		0.9	1.0	1.0	0.9	0.9	0.9	1.0	1.0	0.9	0.9
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
Test substance 0.1	Mean	24.3	106.7	263.3	22.0	8.7	21.0	95.7	255.3	18.3	7.0
	±SD	1.5	3.5	11.7	2.6	1.5	1.0	3.2	5.9	1.5	1.0
Fold Increase		1.0	1 .0	1.0	1.1	0.9	0.9	1.0	1.0	1.0	0.9
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
Test substance 0.32	Mean	25.7	107.0	252.7	19.7	11.0	25.0	104.0	252.7	18.7	10.0
	±SD	2.1	4.6	4.7	2.5	1.0	2.6	3.0	5.1	2.5	1.0
Fold Increase		1.0	1.0	0.9	1.0	1.2	1.0	1.1	1.0	1.0	.1.3
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
Test substance 1	Mean	32.3	118.0	245.7	18.3	13.7	27.0	108.3	241.3	18.0	11.0
	±SD	2.3	4.0	4.5	2.3	1.5	2.6	3.5	4.2	2.0	1.0
Fold Increase		1.3	1.1	.0.9	0.9	1.5	1.1	1.1	0.9	1.0	1.4
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
100 µl of respective Positive Control	Mean	371.7	393.7	593.7	146.3	115.0	355.3	386.7	591.7	135.3	107.0
	±SD	10	11.2	9.7	10.2	9.5	15.5	9.5	15	5.0	5.6
Fold Increase		14.7	3.7	2.2	7.1	12.3	14.6	4.0	2.3	7.4	13.4
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+

Positive controls: With S9 for Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 4 µg/plate of 2-Aminoanthracene. Without S9 for TA98 2 µg/plate of 2-Nitrofluorene; for TA100 and TA1535 1 µg/plate of Sodium azide; for TA102 0.5 µg/plate of Mitomycin C; for TA1537 50 µg/plate of 9-Aminoacridine

Values of Revertants are in Mean ± SD

SUMMARY OF COLONY COUNTS OF REVERTANTS OF EXPERIMENT II - Preincubation Method

Test Concentration  (mg a.i/plate)		No. of Revertants (Mean of 3 Plates)
		With S9					Without S9
		Salmonella typhimurium					Salmonella typhimurium
		TA 98	TA 100	TA 102	TA 1535	TA 1537	TA 98	TA 100	TA 102	TA 1535	TA 1537
100 µL of Distilled water	Mean	30.7	100.3	265.7	20.3	10.3	28.3	101.0	257.3	18.3	9.7
	±SD	2.5	4.0	7.6	3.5	1.5	4.0	4.4	4.2	2.5	1.5
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
Test substance 0.01	Mean	28.3	99.7	280.0	21.3	8.7	26.7	94.3	268.3	20.7	6.7
	±SD	2.1	3.8	8.0	1.5	2.5	1.5	3.1	1.5	1.5	2.1
Fold Increase		0.9	1	1.1	1	0.8	0.9	0.9	1	1.1	0.7
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
Test substance 0.03	Mean	28.0	94.0	277.3	22.0	9.0	24.0	93.3	271.3	19.3	7.7
	±SD	3.0	2.0	2.5	2.0	2.0	2.0	2.1	7.5	1.5	2.1
Fold Increase		0.9	0.9	1.0	1.1	0.9	0.8	0.9	1.1	1.1	0.8
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
Test substance 0.1	Mean	27.7	101.0	258.7	22.3	8.7	26.7	95.7	261.3	18.3	7.0
	±SD	1.5	2.0	6.5	1.2	1.5	0.6	3.2	6.7	2.1	2.6
Fold Increase		0.9	1.0	1.0	1.1	0.8	0.9	0.9	1.0	1.0	0.7
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
Test substance 0.32	Mean	27.7	102.0	262.7	21.3	8.7	28.0	98.0	261.7	21.0	8.3
	±SD	2.1	6.0	8.3	2.1	1.2	3.6	3.6	9.0	3.0	1.2
Fold Increase		0.9	1.0	1.0	1.0	0.8	1.0	1.0	1.0	1.1	0.9
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
Test substance 1	Mean	35.7	115.7	246.3	26.0	14.3	28.0	109.0	245.7	22.3	10.7
	±SD	2.5	7.4	4.0	1.0	1.5	2.0	2.6	3.1	1.5	1.5
Fold Increase		1.2	1.2	0.9	1.3	1.4	1.0	1.1	1.0	1.2	1.1
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+
100 µL of respective Positive Control	Mean	380.3	381.7	599.7	150.3	118.0	353.7	381.7	593.3	142.7	111.7
	±SD	8.5	10.1	12.0	3.5	8.2	12.5	8.0	12.0	12.7	8.7
Fold Increase		12.4	3.8	2.3	7.4	11.4	12.5	3.8	2.3	7.8	11.6
Lawn Intensity		4+	4+	4+	4+	4+	4+	4+	4+	4+	4+

Positive controls: With S9 for Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 4 µg/plate of 2-Aminoanthracene. Without S9 for TA98 2 µg/plate of 2-Nitrofluorene; for TA100 and TA1535 1 µg/plate of Sodium azide; for TA1020.5µg/plate of Mitomycin C; for TA1537 50 µg/plate of 9-Aminoacridine

Values of Revertants are in Mean±SD

CYTOTOXICITY TEST

Test Item Concentration (mg a.i/plate)	No. of revertants/plate
	with S9						without S9
	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity
Vehicle Control	91	98	105	98	7	4+	94	101	90	95	5.6	4+
0.1	97	94	104	98	5.1	4+	91	98	101	97	5.1	4+
0.2	95	101	98	98	3	4+	94	100	93	96	3.8	4+
0.3	96	93	102	97	4.6	4+	92	101	96	96	4.5	4+
0.4	91	102	103	99	6.7	4+	92	93	95	93	1.5	4+
0.5	105	98	96	100	4.7	4+	97	95	91	94	3.1	4+
0.6	103	101	99	101	2	4+	97	93	98	96	2.6	4+
0.7	102	97	102	100	2.9	4+	97	91	102	97	5.5	4+
0.8	101	95	92	96	4.6	4+	92	96	98	95	3.1	4+
0.9	89	97	93	93	4	4+	87	90	92	90	2.5	4+
1	86	84	89	86	2.5	4+	85	81	86	84	2.6	4+

Values of Revertants are in Mean±SD

Lawn intensity:

4+= Thick lawn: Distinguished by a healthy (Normal) background lawn comparable tovehiclecontrol plates.    

Conclusions:

Based on the results of the study, it is concluded that test item is “non-mutagenic” in the bacterial reverse mutation test up to the highest tested concentration of 1 mg a.i/plate under test conditions.

Executive summary:

The test item was evaluated for mutagenicity in bacterial reverse mutation test, according to OECD guideline 471 (1997).

The test item was found soluble in distilled water at a concentration of 50 mg a.i/ml and resulted in heavy precipitation at 3, 4 and 5 mg a.i/plate; thus, on the basis of test item solubility and precipitation tests results, the initial cytotoxicity test was performed at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg a.i/plate. Initial cytotoxicity test was performed with Salmonella typhimurium TA100 tester strain both in the presence and absence of metabolic activation system.

On the basis of cytotoxicity results 1 mg a.i/plate was considered as the highest test concentration for mutation assay by comparing with the vehicle control.

Two independent trials (trial 1 and 2) were conducted by plate incorporation method (standard metabolic activation system) and pre incubation method (reductive metabolic activation system), in the presence and absence of metabolic activation system at concentrations of 0.01, 0.03, 0.10, 0.32 and 1 mg a.i/plate.

Vehicle control (distilled water) and the appropriate positive controls (2 -nitrofluorene, sodium azide and 9 -aminoacridine, mitomycin C for trials “without metabolic activation” and 2 -aminoanthracene for trials “with metabolic activation”) were tested simultaneously.

Tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537.

Based on the experimental results obtained, the mean numbers of revertant colonies at tested concentrations were comparable to those of the vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials.

The number of revertant colonies in the positive controls resulted in 2.2 to 14.7 fold increase under identical conditions.

Conclusion

Based on test results, it is concluded that test item is “non-mutagenic” in the bacterial reverse mutation test up to the highest tested concentration of 1 mg a.i/plate under test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test item was evaluated for mutagenicity in bacterial reverse mutation test, according to OECD guideline 471 (1997).

Based on solubility and precipitation tests, the initial cytotoxicity test was performed at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg a.i/plate on TA100 tester strain both in the presence and absence of metabolic activation system.

On the basis of cytotoxicity results, 1 mg a.i/plate was considered as the highest test concentration for mutation assay by comparing with the vehicle control.

Two independent trials (trial 1 and 2) were conducted by plate incorporation method (standard metabolic activation system) and pre incubation method (reductive metabolic activation system), in the presence and absence of metabolic activation system at concentrations of 0.01, 0.03, 0.10, 0.32 and 1 mg a.i/plate.

Tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537.

There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both trials.

Positive controls were run and were valid.

Justification for classification or non-classification

The CLP Regulation (EC) No 1272/2008 reports hazard categories for classification of germ cell mutagens.

CATEGORY 1:

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

Category 1A:

The classification in Category 1A is based on positive evidence from human epidemiological studies. Substances to be regarded as if they induce heritable mutations in the germ cells of humans.

Category 1B:

The classification in Category 1B is based on: — positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; or — positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells. It is possible to derive this supporting evidence from mutagenicity/genotoxicity tests in germ cells in vivo, or by demonstrating the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells; or — positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

CATEGORY 2:

Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans The classification in Category 2 is based on: — positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from: — somatic cell mutagenicity tests in vivo, in mammals; or — other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays. Note: substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

A bacterial reverse mutation assay is considered as a screening test for genotoxicity. A positive response would imply further testing to assess the genotoxic potential of a substance.

Acid Blue 193 was found to be non-mutagenic under test conditions, thus no classification applies.