Disodium ar,ar'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[(4-chlorophenoxy)benzenesulphonate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 08 to June 15, 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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acid Green 040

IUPAC Name:

Acid Green 040

Method

Species / strain

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.
- Storage of the Test System: stock cultures of tester strains 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.
- Bacteria growth: each of the tester strain from the master plates were grown in 10 ml of oxoid nutrient broth no.2. A fresh culture of bacteria was grown up to late exponential or early stationary phase of growth. The flask with 10 ml of oxoid nutrient broth no.2 and bacterial inoculum was incubated in a water bath at 37 ± 1 ºC for approximately 15 hours. The inoculum was adjusted to a density of 18 × 10^8 cells/ml.

CHARCATERIZATION OF TESTER STRAINS
After preparation of the master plates, the growth requirements and the genetic identity of Salmonella typhimurium strains like histidine requirement, sensitivity to UV radiation, resistance of strains TA98, TA100 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:

rat liver S9 fraction

Test concentrations with justification for top dose:

Main experiment: 0.008, 0.03, 0.08, 0.3 and 0.8 mg a.i/plate, with and without metabolic activation system
Cytotoxicity: vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg a.i/plate, with and without metabolic activation system

Vehicle / solvent:

- Solvent used: DMSO

Controls

Negative solvent / vehicle 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
Plate Incorporation Method: Trial I
- Plate composition: test item, vehicle, positive control and the tester strains along with S9/phosphate buffer saline were mixed with 2 ml soft agar and poured on to minimal glucose agar plates.
- Bacterial culture: containing approximately 10^8 viable cells.
- Incubation: plates were incubated at 37 ± 1 °C for 48 hours and 30 minutes.

Preincubation Method: Trial II
- Pre-incubation: test item, vehicle, positive control and the tester strains along with S9/phosphate buffer saline were mixed and incubated in a shaker for 30 minutes.
- Plate composition for post-incubation: after incubation, the test constituents were mixed with 2 ml soft agar and poured on to minimal glucose agar plates.
- Bacterial culture: containing approximately 10^8 viable cells.
- Post-incubation: plates were incubated at 37 ± 1 °C for 65 hours.

NUMBER OF REPLICATIONS: triplicate plates for five treatment concentrations, vehicle control and positive control were maintained in each experiment.

OBSERVATIONS
During both the experiments, the revertant colonies for each strain within the test item dilution series were counted manually.

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 41 hours at 37 ± 1 ºC for the plate incorporation method and the pre incubation method.
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.

COUNTING OF REVERTANT COLONIES
Revertant colonies for each respective strain, within the test item dilution series were counted manually.

VIABLE COUNT
Viable count for the different bacterial strains on nutrient agar plates was counted manually.

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

PRECIPITATION TEST
The stock solution of test item was serially diluted and plated to get concentrations of 1, 2, 3, 4 and 5 µl/plate using Dimethyl sulphoxide. A quantity of 100 µl of each concentration was separately mixed with 2 ml of molten soft agar, vortexed and spread onto minimal glucose agar plates. Plates were incubated for 2 hours at 37 ± 1 °C.

CYTOTOXICITY TEST
Salmonella typhimurium TA100 tester strain was exposed to test item concentrations, both in the presence and absence of metabolic activation, along with the concurrent vehicle control (dimethyl sulphoxide). Each concentration, including control, was tested in triplicate.
Each concentration was mixed with soft agar containing histidine and biotin, S9 mix (for presence of metabolic activation), phosphate buffer saline (for absence of metabolic activation), Salmonella typhimurium TA100 (cell density approximately 18 ×10^8 cells/ml). The mixture was added to the overlay of properly labeled minimal glucose agar plates. The plates were incubated at 37 ± 1 ºC for 48 hours and 8 minutes.

PREPARATION OF S9 HOMOGENATE
Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate were 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 mg/ml 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 incubating it 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-Aminoantracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 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 pH 7.28 for initial cytotoxicity and pH 7.30 for plate incorporation and for preincubation method to get the concentration of 10 % (v/v).

CRITERIA FOR ACCEPTABILITY OF THE TEST
The mutation test is considered acceptable as it met 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 will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

slightly reduced lawn when compared to vehicle control to the highest concentration

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Plate Incorporation Method: Trial I
All the tester strains treated with the test item, at the concentration of 0.8 mg a.i/plate showed slightly reduced lawn (grade 3+) when compared to vehicle control and showed reduction in mean number of revertant colonies/plate and bacterial background lawn when compared to the vehicle control.
All the tester strains treated with the test item, at the concentrations 0.008, 0.03, 0.08, 0.3 mg a.i/plate 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 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 the vehicle control.
The specific positive controls tested simultaneously produced approximately 2.3 to 14.7 fold increase in mean number of revertants as compared to the vehicle control.

Preincubation Method: Trial II
All the tester strains treated with the test item, at the concentration of 0.8 mg a.i/plate showed slightly reduced lawn (grade 3+) when compared to vehicle control and showed reduction in mean number of revertant colonies/plate and bacterial background lawn when compared to the vehicle control.
All the tester strains treated with test item, at the concentrations tested 0.008, 0.03, 0.08, 0.3 mg a.i/plate 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 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 the vehicle control.
The specific positive controls tested simultaneously produced approximately 2.3 to 13.8 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 pre incubation method each tester strain resulted in acceptable range of 1×10^9 to 2×10^9 CFU/ml.

QUALITY CONTROL PLATES
Control plates incubated at 37 ± 1 °C for 66 hours and 30 minutes for plate incorporation method and for pre incubation method were checked for contamination. No microbial contamination was observed.

CYTOTOXICITY TEST
Cytotoxicity was observed by a thinning of the bacterial background lawn. The strain exposed with test item, in the presence and absence of metabolic activation system, caused cytotoxicity: absent lawn at 4 and 5 mg a.i/plate extremely reduced lawn at 3 mg a.i/plate, moderately reduced lawn at 0.9, 1 and 2 mg a.i/plate and slightly reduced lawn at 0.8 mg a.i/plate and thick lawn at 0.5, 0.6, 0.7 mg a.i/plate when compared to vehicle control.
On the basis of cytotoxicity results, 0.8 mg a.i/plate was considered as the highest test concentration for mutation assay.

PRECIPITATION TEST
The test item resulted in no precipitation at and up to 5 mg/plate tested concentrations.

SOLUBILITY TEST
The test item was found to be soluble in dimethyl sulphoxide at a concentration of 50 mg a.i/ml (purity correction factor considered).

CRITERIA FOR ACCEPTABILITY OF THE TEST

Any other information on results incl. tables

Plate Incorporation Method

Treatment	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
Vehicle Control	100 µl DMSO	Mean	27.3	95	267	22.3	10.7		24.7	91.7	258.3	20.7	8.3
		±SD	2.5	6.2	8	1.5	1.2		3.1	3.1	10.2	3.1	2.5
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Test item	0.008	Mean	22.7	98.7	260	23.7	7		21.3	98.3	261.3	19.7	6
		±SD	2.1	2.5	9.8	1.2	2.6		1.5	10.4	9.1	3.2	1
	Fold Increase		0.8	1	1	1.1	0.7		0.9	1.1	1	1	0.7
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.03	Mean	23	95.3	262.3	21	9		21	93	258.3	18.7	7.3
		±SD	3	7.5	8	2	2.6		2	4	3.5	1.5	2.5
	Fold Increase		0.8	1	1	0.9	0.8		0.9	1	1	0.9	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.08	Mean	24.7	95.7	267.3	19.7	7		21.7	92.3	257	19	6.7
		±SD	2.1	5.5	10.7	2.1	2		3.5	5.1	4	2.6	3.1
	Fold Increase		0.9	1	1	0.9	0.7		0.9	1	1	0.9	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.3	Mean	18.7	85.3	260.7	17	4.7		17.3	84.3	253	15.3	4.3
		±SD	1.5	2.5	8.7	1	1.2		1.2	2.5	10.5	1.5	0.6
	Fold Increase		0.7	0.9	1	0.8	0.4		0.7	0.9	1	0.7	0.5
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.8	Mean	13.3	61	209	14.3	3		13.7	55.3	207	13	2.7
		±SD	2.1	2	6.6	0.6	1		1.5	2.5	6	1	0.6
	Fold Increase		0.5	0.6	0.8	0.6	0.3		0.6	0.6	0.8	0.6	0.3
	Lawn Intensity		3+	3+	3+	3+	3+		3+	3+	3+	3+	3+
Positive Controls	100 µl	Mean	371	377.3	604.7	147.3	116.7		363	383.7	583.3	144.7	113
		±SD	10.5	12.7	12.6	13.6	8		13.1	21.1	13.5	7.4	6.6
	Fold Increase		13.6	4	2.3	6.6	10.9		14.7	4.2	2.3	7	13.6
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Positive controls:

For with S9:       

For Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

For 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

Preincubation Method

Treatment	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
Vehicle Control	100 µl DMSO	Mean	28.3	105.3	256.7	20.7	11.3		26.7	99.3	258.7	21.7	8.3
		±SD	3.5	3.8	8.1	2.5	1.5		3.5	4.2	7.4	1.5	2.1
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Test item	0.008	Mean	25.7	101.3	259	19.3	9.7		21	96.7	258	19	6
		±SD	3.5	3.1	6	2.5	2.1		2.6	2.5	3.6	2.6	1
	Fold Increase		0.9	1	1	0.9	0.9		0.8	1	1	0.9	0.7
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.03	Mean	26.7	99.7	264.7	20.7	11.3		26	93.7	258.3	19.3	8
		±SD	5	4.2	8.4	3.1	2.5		1	2.1	5.5	3.1	2
	Fold Increase		0.9	0.9	1	1	1		1	0.9	1	0.9	1
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.08	Mean	24	96.7	268.3	21	8.7		20.7	95.3	255.3	18	8.7
		±SD	2.6	6.8	4.5	2.6	2.1		3.1	2.1	5.5	2.6	3.5
	Fold Increase		0.8	0.9	1	1	0.8		0.8	1	1	0.8	1
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.3	Mean	21.3	85.3	239.7	16.3	5.3		19.3	84.7	235.3	15.7	5
		±SD	1.5	3.1	7.1	1.5	1.2		1.5	4.5	4.5	1.5	2
	Fold Increase		0.8	0.8	0.9	0.8	0.5		0.7	0.9	0.9	0.7	0.6
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0..8	Mean	16.3	62.3	209	14.7	4.3		15.3	55	206	13.7	3
		±SD	1.5	4.2	6	1.5	0.6		1.5	3	5.6	1.5	1
	Fold Increase		0.6	0.6	0.8	0.7	0.4		0.6	0.6	0.8	0.6	0.4
	Lawn Intensity		3+	3+	3+	3+	3+		3+	3+	3+	3+	3+
Positive Controls	100 µl	Mean	379.7	402.3	594	138.7	111.7		363	390.3	592.7	146.7	114.7
		±SD	12.5	10	12.8	13.1	9.1		11.1	4.5	7.6	19.8	7.8
	Fold Increase		13.4	3.8	2.3	6.7	9.9		13.6	3.9	2.3	6.8	13.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Positive controls:

For with S9:       

For Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

For 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

Applicant's summary and conclusion

Conclusions:

Based on the results obtained from the study, it can be concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.8 mg a.i/plate under the tested conditions.

Executive summary:

The test item was evaluated for mutagenicity in a Bacterial Reverse Mutation Test, according to the OECD guideline for testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

The test item found to be soluble in dimethyl sulphoxide at a concentration of 50 mg a.i/ml. Test item resulted in no precipitation at and up to 5 mg a.i/plate. Based on pretest results, the highest concentration selected for initial cytotoxicity test was 5 mg a.i/plate.

Salmonella typhimurium TA100 strain was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg a.i/plate. The test item resulted in cytotoxicity at 0.8, 0.9, 1, 2, 3, 4 and 5 mg a.i/plate tested concentration in initial cytotoxicity test. Based on the results of initial cytotoxicity test, concentrations of 0.008, 0.03, 0.08, 0.3 and 0.8 mg a.i/plate were selected for testing in the mutation test by plate incorporation method.

The test item was assessed for its mutagenic effects using Salmonella typhimurium strains TA98, TA100, TA 102, TA1535, and TA1537. The test item was tested at the concentrations of 0.008, 0.03, 0.08, 0.3 and 0.8 mg a.i/plate for plate incorporation method and for preincubation method using Dimethyl sulphoxide as vehicle based on the results of solubility, precipitation and initial cytotoxicity test. The study was conducted with and without metabolic activation (S9 fraction). The S9 fraction was prepared from sodium phenobarbitone and β-naphthoflavone induced rat liver. Vehicle control and appropriate positive controls (2-nitrofluorene, sodium azide, 9-Aminoacridine and Mitomycin C, for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. Two trials were carried out for the study in triplicate. Trial I was carried out as plate incorporation method and trial II as pre incubation method.

All the tester strains treated with the test item at the concentration of 0.8 mg a.i/plate showed slightly reduced lawn (grade 3+) and a reduction in mean number of revertant colonies/plate in the presence and absence of metabolic activation when compared to the vehicle control. No inhibitory effects of the test item were observed at the concentration ≤ 0.3 mg a.i /plate.

There was no substantial increase in number of revertant colonies at 0.008, 0.03, 0.08 and 0.3 mg a.i/plate of the tested concentrations in both the trials.

The number of revertant colonies in the positive controls resulted in 2.3 to 14.7 fold increase under identical conditions.

Conclusion

Based on the results obtained from the study, it can be concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.8 mg a.i/plate under the tested conditions.