1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

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:

26 October - 12 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st 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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, June 2012

Version / remarks:

June 2012

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:

1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

EC Number:

235-521-4

EC Name:

1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Cas Number:

12262-32-7

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)- with sodium polysulfide, leuco derivatives

Test material form:

solid

Specific details on test material used for the study:

Date of production: 26.03.2015
Expiration date: 26.03.2020

Method

Target gene:

his/trp
Genotypes of the Strains Used for Mutagenicity Testing:
In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and beta-naphthoflovone induce rat liver, S9-mix

Test concentrations with justification for top dose:

1600, 500, 160, 50, 16, 5, and 1.6 µg/plate

Vehicle / solvent:

Vehicle: dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: DMSO was found as appropriate vehicle for preparing the test item solutions. Based on the laboratory’s historical control database, this vehicle is compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine

Remarks:

TA98, without S9 mix, 4 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535, without S9 mx, 2 µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537, without S9 mix, 50 µg/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.coli WP2, without S9 mix, 2 µL/plate

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All Salmonella strains tested, with S9 mix, 2 µg/plate; E.coli WP2, with S9 mix, 50 µg/plate

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony count, background lawn development

Rationale for test conditions:

Justification of concentrations:
Selection of the concentration range was done on the basis of a Solubility Test and a Concentration Range Finding Test.
Based on the solubility test, a stock solution (suspension) with a nominal concentration of 50 mg/mL was prepared in DMSO and diluted in 6 steps by factor of approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) was determined at the concentrations of 5000; 1600; 500; 160; 50; 16 and 5 µg/plate. In the informatory experiment the revertant colony numbers of vehicle control plates with and without S9 Mix were in line the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.

Evaluation criteria:

The colony numbers on the untreated, vehicle control, positive control and the test item treated plates were determined visually by manual counting, vs control

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.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
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.

Statistics:

The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Validity of the Performed Experiments
The tester strains used in this study 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 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 dimethyl sulfoxide (DMSO) 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.

Controls
In the performed Initial and Confirmatory Mutation Test multiple test items were tested with reference values from the common parallel controls. In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the dimethyl sulfoxide (DMSO) 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. The revertant colony numbers of the untreated and ultrapure water control plates in different experimental phases were slightly higher or lower than the DMSO 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.

Initial and Confirmatory Mutation Tests (Plate Incorporation Test and Pre-Incubation Test)
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with C. I. Leuco Sulfur Green 2 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 Initial Mutation Test (Plate Incorporation Test) in S. typhimurium TA98 strain at 50 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 1.63, which was far below the genotoxicological threshold for being positive.
In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was observed. In the Initial Mutation Test inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain only, in the absence and also in the presence of exogenous metabolic activation. In the Confirmatory Mutation Test unequivocal inhibition was noticed in all examined Salmonella typhimurium strains , in the absence of exogenous metabolic activation. The cytotoxicity was indicated by decreased revertant colony counts (some of them below the corresponding historical control data ranges) and/or affected background lawn development: reduced or slightly reduced background lawn. For the cytotoxicity tendency (it did not show a clear dose-dependent tendency), the effect of precipitate formation was supposed. The table contains the unequivocal results where the obtained revertant colony numbers were below the vehicle (in some cases below the corresponding historical control data ranges), and/or affected background lawn development occurred.

All of the further observed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system. In general, 160 µg/plate was considered as lowest concentration showing unequivocal cytotoxicity. When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentration of 1600 µg/plate in absence and at 1600 and 500 µg/plate in the presence of S9 following the plate incorporation procedure and in the absence and presence of S9 following the pre-incubation procedure. For confirmation of manual evaluations (made by naked eye) all of the plates were checked for colony and background lawn development by microscope at 40X magnification. At this magnification further test item precipitate was not noticed at lower concentration levels.

Any other information on results incl. tables

Table 4: Summary of the Results of the Concentration Range Finding Test

Concentration Range Finding Test (Informatory Toxicity Test)
Concentrations (µg/plate)	Salmonella typhimuriumtester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	24.3	1.28	17.7	0.83	97.3	1.15	115.3	1.07
DMSO Control	19.0	1.00	21.3	1.00	84.7	1.00	107.3	1.00
Ultrapure Water Control	–	–	–	–	100.7	1.00	–	–
5000	#	#	#	#	75.0	0.89	101.3	0.94
1600	18.3	0.96	16.7	0.78	98.0	1.16	99.3	0.93
500	19.7	1.04	14.7	0.69	97.7	1.15	112.3	1.05
160	18.7	0.98	18.7	0.88	84.7	1.00	102.7	0.96
50	16.0	0.84	22.7	1.06	99.3	1.17	95.7	0.89
16	25.0	1.32	21.0	0.98	88.7	1.05	105.0	0.98
5	27.7	1.46	17.7	0.83	94.7	1.12	102.7	0.96
NPD (4 µg/plate)	236.7	12.46	–	–	–	–	–	–
SAZ (2 µg/plate)	–	–	–	–	1093.3	10.86	–	–
2AA (2 µg/plate)	–	–	850.7	39.88	–	–	2075.3	19.34

MR: Mutation Rate                                   #   : the revertants could not be counted, evaluated

NPD: 4-Nitro-1,2-phenylenediamine

SAZ: Sodium azide

2AA: 2-aminoanthracene

Table 5: Summary of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (µg/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	21.7	1.33	26.0	0.88	86.0	1.01	117.3	1.27	9.3	0.88	11.0	0.94	7.3	1.29	4.7	0.64	18.3	0.83	19.3	0.94
DMSO Control	16.3	1.00	29.7	1.00	85.0	1.00	92.3	1.00	10.7	1.00	11.7	1.00	5.7	1.00	7.3	1.00	22.0	1.00	20.7	1.00
Ultrapure Water Control	–	–	–	–	102.7	1.00	–	–	12.7	1.00	–	–	–	–	–	–	19.7	1.00	–	–
1600	19.7	1.20	18.7	0.63	111.3	1.31	106.7	1.16	7.3	0.69	8.3	0.71	0.3	0.06	3.3	0.45	15.7	0.71	21.7	1.05
500	18.0	1.10	25.7	0.87	102.3	1.20	129.0	1.40	12.3	1.16	9.3	0.80	2.0	0.35	10.0	1.36	15.0	0.68	22.3	1.08
160	25.7	1.57	24.0	0.81	102.7	1.21	135.0	1.46	9.3	0.88	10.7	0.91	5.3	0.94	9.0	1.23	18.3	0.83	18.7	0.90
50	26.7	1.63	20.3	0.69	95.7	1.13	121.0	1.31	9.3	0.88	15.3	1.31	5.7	1.00	7.3	1.00	12.0	0.55	21.0	1.02
16	19.7	1.20	19.7	0.66	98.0	1.15	127.0	1.38	9.0	0.84	16.3	1.40	6.0	1.06	6.7	0.91	19.7	0.89	28.3	1.37
5	20.0	1.22	17.3	0.58	96.0	1.13	114.3	1.24	13.7	1.28	16.3	1.40	7.0	1.24	6.3	0.86	14.7	0.67	19.3	0.94
1.6	18.0	1.10	16.7	0.56	98.3	1.16	101.3	1.10	7.7	0.72	14.0	1.20	6.0	1.06	8.7	1.18	15.7	0.71	22.3	1.08
NPD (4 µg/plate)	274.7	16.82	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 µg/plate)	–	–	–	–	1240.0	12.08	–	–	561.7	44.34	–	–	–	–	–	–	–	–	–	–
9AA (50 µg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	597.3	105.41	–	–	–	–	–	–
MMS (2 µL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	892.0	45.36	–	–
2AA (2 µg/plate)	–	–	2336.0	78.74	–	–	2317.3	25.10	–	–	183.7	15.74	–	–	169.7	23.14	–	–	–	–
2AA (50 µg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	220.0	10.65

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

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

Table 6: Summary of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (µg/plate)	Salmonella typhimuriumtester 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	18.0	0.95	17.7	0.98	97.3	1.20	109.7	1.02	10.7	1.23	12.0	0.97	13.7	1.00	5.7	0.81	18.0	0.93	30.0	1.22
DMSO Control	19.0	1.00	18.0	1.00	81.3	1.00	108.0	1.00	8.7	1.00	12.3	1.00	13.7	1.00	7.0	1.00	19.3	1.00	24.7	1.00
Ultrapure Water Control	–	–	–	–	90.3	1.00	–	–	14.3	1.00	–	–	–	–	–	–	22.3	1.00	–	–
1600	16.0	0.84	17.0	0.94	104.7	1.29	97.3	0.90	5.3	0.62	12.0	0.97	1.0	0.07	8.0	1.14	21.3	1.10	21.0	0.85
500	11.7	0.61	17.7	0.98	51.0	0.63	112.7	1.04	4.3	0.50	11.7	0.95	1.3	0.10	7.3	1.05	21.3	1.10	24.3	0.99
160	9.7	0.51	23.3	1.30	90.7	1.11	114.3	1.06	2.7	0.31	10.7	0.86	5.0	0.37	8.3	1.19	20.7	1.07	22.0	0.89
50	30.0	1.58	20.0	1.11	102.0	1.25	101.0	0.94	8.0	0.92	13.0	1.05	6.7	0.49	6.7	0.95	22.0	1.14	25.7	1.04
16	21.7	1.14	15.7	0.87	77.3	0.95	115.3	1.07	7.0	0.81	9.3	0.76	13.7	1.00	8.0	1.14	14.7	0.76	27.3	1.11
5	18.0	0.95	18.0	1.00	90.0	1.11	114.3	1.06	8.3	0.96	12.0	0.97	12.7	0.93	9.0	1.29	21.0	1.09	31.7	1.28
1.6	17.3	0.91	21.7	1.20	88.7	1.09	105.0	0.97	10.0	1.15	10.3	0.84	13.0	0.95	10.0	1.43	18.7	0.97	34.7	1.41
NPD (4 µg/plate)	188.7	9.93	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 µg/plate)	–	–	–	–	1248.0	13.82	–	–	794.7	55.44	–	–	–	–	–	–	–	–	–	–
9AA (50 µg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	264.0	19.32	–	–	–	–	–	–
MMS (2 µL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	824.0	36.90	–	–
2AA (2 µg/plate)	–	–	684.0	38.00	–	–	984.0	9.11	–	–	160.0	12.97	–	–	93.3	13.33	–	–	–	–
2AA (50 µg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	146.7	5.95

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

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

.

Table 7: Historical Control Values for Revertants/Plate (for the Period of 2008-2015)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.4	106.0	10.4	8.1	25.6
		SD	3.7	27.3	1.5	2.5	5.5
		Minimum	9	65	3	2	11
		Maximum	39	157	23	19	45
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	28.0	117.1	11.9	9.0	34.3
		SD	4.2	19.4	1.5	2.0	5.4
		Minimum	12	75	4	3	18
		Maximum	48	166	24	20	56
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	20.9	101.4	10.3	7.9	24.9
		SD	3.5	26.2	1.4	2.5	4.9
		Minimum	10	65	3	2	11
		Maximum	39	150	23	20	44
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.1	114.7	12.0	8.8	34.2
		SD	4.0	19.3	1.5	2.1	5.2
		Minimum	15	71	4	3	16
		Maximum	48	161	24	20	56
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	22.4	105.5	10.4	7.5	26.3
		SD	3.6	27.6	1.6	2.3	5.9
		Minimum	12	67	3	2	13
		Maximum	36	156	24	15	47
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	28.0	117.4	11.5	8.7	35.2
		SD	4.0	19.8	1.4	2.3	5.2
		Minimum	15	83	4	4	18
		Maximum	43	166	22	16	56
			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	255.6	958.9	842.1	467.4	712.3
		SD	30.7	149.9	134.0	105.7	57.5
		Minimum	123	522	354	109	320
		Maximum	647	1927	1871	1498	1283
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1224.8	1431.9	165.4	148.0	264.7
		SD	293.8	339.9	35.1	21.3	74.2
		Minimum	409	581	85	68	141
		Maximum	2587	2923	507	407	487

Abbreviations:    TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2uvrA

                               SD: Standard deviation; DMSO: Dimethyl sulfoxide

Applicant's summary and conclusion

Conclusions:

In the bacterial reverse mutation assay (Ames test) according to OECD guideline 471 the test item did not induce gene mutations.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (Ames test) according to the OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 mix) prepared from livers of phenobarbital/beta-naphthoflavone-induced rats. 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 Solubility Test and the Concentration Range Finding Test the test item was dissolved in dimethyl sulfoxide (DMSO). Based on the results of the preliminary Concentration Range Finding Test (Informatory Toxicity Test) the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:1600; 500; 160; 50; 16; 5 and 1.6 µg/plate.

In the main experiments (when evaluated by naked eye) test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentration of 1600 µg/plate in absence and at 1600 and 500 µg/plate in the presence of S9 following the plate incorporation procedure and in the absence and presence of S9 following the pre-incubation procedure. The obtained precipitate did not interfere with the scoring of the colonies in any case. In the Initial Mutation Test inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain in the absence and also in the presence of exogenous metabolic activation. In the Confirmatory Mutation Test unequivocal inhibition was noticed in all examined Salmonella typhimurium strains, in the absence of exogenous metabolic activation. The inhibitory effect was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and affected background lawn development: reduced or slightly reduced background lawn. In general, 160 µg/plate was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO) 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 coloniesand the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive controlin all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with 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 by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.