Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

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:

17 July 2017 - 29 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

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:

Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

EC Number:

288-914-8

EC Name:

Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

Cas Number:

85940-25-6

Molecular formula:

Molecular formula is not available

IUPAC Name:

Reaction product of phenol, 4-​(9H-​carbazol-​3-​ylamino)​- with sodium polysulfide, leuco derivatives

Test material form:

solid: particulate/powder

Details on test material:

Test Item: Leuco Sulfur Blue 20P
Appearance: Black powder

Method

Target gene:

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 (WP2 uvrA) 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:

post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/-naphthoflavone-induced rats.

Test concentrations with justification for top dose:

1600, 1000, 500, 250, 160, 100, 50, 16 and 5 µg/plate

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

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

Details on test system and experimental conditions:

Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures.

Storage of Tester Strains

The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.

Confirmation of Phenotypes of Tester Strains

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.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.

Spontaneous Reversion of Tester Strains

Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. 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 11-13 hours in a 37 oC Benchtop Incubator Shaker.

Viability and the Cell Count of the Testing Bacterial Cultures

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 System

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Rat Liver S9 Fraction

The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

Rationale for test conditions:

Selection of the concentration range for the initial mutation test was done on the basis of solubility test and concentration range finding test. The investigated concentration range for the confirmatory mutation test was chosen based on the results of the initial mutation test.At the concentration choice the non-toxicityof the test item and the appearance of precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye.To confirm and to investigate the reproducibility of the positive result of the initial mutation test the following nine concentration levels were investigated in the confirmatory mutation test:±S9 mix: 1600, 1000, 500, 250, 160, 100, 50, 16 and 5 µg/plate.
When evaluated by the naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains down to and including the concentration level of 500 µg/plate in the absence and down to and including the concentration level of 250 µg/plate in the presence of exogenous metabolic activation following the plate incorporation procedures.In the initial mutation test an inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain in the absence (in the concentration range of 1600-160 µg/plate) and also in the presence (at the concentrations of 1600 and 500 µg/plate) of exogenous metabolic activation. In confirmatory mutation test the inhibitory effect was indicated by absent or decreased revertant colony counts and/or 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 solvent control (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.

Evaluation criteria:

The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined visually by manual counting, and the mean values, standard deviations and the mutation rates were calculated.
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 solvent 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 solvent 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 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In general, 160 µg/plate was considered as lowest concentration showing cytotoxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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 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 solvent control in all 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 the tester strains. The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates showed characteristic mean numbers agreed with the actual historical control data ranges in the examined strains in both main experimental phases. Seven concentration levels were investigated in the initial mutation test and nine in the confirmatory mutation test. In the performed main 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) solvent 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 Salmonella typhimurium TA98 the revertant colony numbers of 2-aminoanthracene (2AA) were above the corresponding historical control data range; however the higher counts were considered as acceptable without any effect on the final conclusion of the study. 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, solvent and positive controls were in line with the criteria for validity of the assay.

Initial Mutation Test (Plate Incorporation Test)

In this test negative mutagenicity results were obtained in Salmonella typhimurium TA98, TA100, TA1535 and Escherichia coli WP2 uvrA strains in the absence and also in the presence of exogenous metabolic activation (±S9 mix). Unequivocal positive results were noticed following treatment with the test item in the investigated Salmonella typhimurium TA1537 strain (±S9 mix). The obtained revertant colony number increases were clearly above the corresponding historical control data range and the relevant genotoxicological threshold for being positive at the concentrations of 50, 16 and 5 µg/plate (-S9 mix) and at the concentration range of 1600-16 µg/plate (+S9 mix) (with exception of the concentration level of 500 µg/plate, where the obtained increase was above the historical control data range, but remained below the threshold for being positive). The higher revertant colony numbers (when compared to the revertant colony numbers of the solvent control) were above the corresponding historical control data range; however remained below the threshold, for being positive in S. typhimurium TA98 at the concentration range of 1600-50 µg/plate (-S9 mix). Under the experimental conditions applied, the test item induced gene mutations by frameshifts in the genome of the Salmonella typhimurium TA1537 tester strain examined. Inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain in the absence (in the concentration range of 1600-160 µg/plate) and also in the presence (at the concentrations of 1600 and 500 µg/plate) of exogenous metabolic activation. The inhibitory effect was indicated by absent or decreased revertant colony counts (compared to the revertant colony numbers of the DMSO control) and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 160 µg/plate was considered as lowest concentration showing cytotoxicity. When evaluated by the naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at 1600 and 500 µg/plate in the absence and also in the presence of exogenous metabolic activation. To confirm and to investigate the reproducibility of this positive result a confirmatory mutation test was performed with S. typhimurium TA1537 in the absence and presence of exogenous metabolic activation (±S9 mix). The strains Salmonella typhimurium TA98, TA100, TA1535 and Escherichia coli WP2 uvrA were not further investigated.

Confirmatory Mutation Test (Plate Incorporation Test)

The positive results already noticed in the initial mutation test in the investigated Salmonella typhimurium TA1537 strain (±S9 mix) were successfully confirmed. The revertant colony number increases were above the corresponding historical control data range and the relevant genotoxicological threshold for being positive at the concentrations of 100, 50, 16 and 5 µg/plate (-S9 mix) and at the concentration range of 500-50 µg/plate (+S9 mix). The revertant colony number increase was above the historical control data range and followed a dose-relationship, but remained below the threshold for being positive at 16 µg/plate (+S9 mix). All of the obtained increased tendencies followed clear dose-relationship. The inhibitory tendencies were similar to that already observed in the initial mutation test. An inhibitory effect of the test item was observed in the concentration range of 1600-160 µg/plate in the absence and at the concentrations of 1600, 1000 and 500 µg/plate in the presence of exogenous metabolic activation. The inhibitory effect was indicated by decreased revertant colony counts (compared to the revertant colony numbers of the DMSO control) and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 160 µg/plate was considered as lowest concentration showing cytotoxicity. Non-interfering test item precipitate was noticed on the plates at 1600, 1000 and 500 µg/plate in the absence and at 1600-250 µg/plate, in the presence of exogenous metabolic activation.

Any other information on results incl. tables

Summary Table of the Results of the Concentration Range Finding Test

Concentration 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 Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	16.3	0.92	18.3	0.82	85.7	0.94	120.0	1.10
DMSO Control	17.7	1.00	22.3	1.00	90.7	1.00	108.7	1.00
Ultrapure Water Control	–	–	–	–	86.0	1.00	–	–
5000	20.3	1.15	13.7	0.61	85.3	0.94	88.7	0.82
1600	22.0	1.25	18.7	0.84	93.0	1.03	94.0	0.87
500	16.3	0.92	23.7	1.06	110.0	1.21	105.3	0.97
160	13.0	0.74	26.7	1.19	97.7	1.08	119.7	1.10
50	14.3	0.81	29.3	1.31	96.7	1.07	113.0	1.04
16	20.0	1.13	36.7	1.64	99.7	1.10	101.7	0.94
5	20.0	1.13	33.7	1.51	92.7	1.02	104.0	0.96
NPD (4mg)	193.3	10.94	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	776.0	9.02	–	–
2AA (2mg)	–	–	1066.7	47.76	–	–	2017.3	18.56

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

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

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	17.3	0.96	23.3	0.93	88.7	1.08	110.7	1.09	9.7	1.00	12.3	0.97	6.3	0.90	7.0	0.84	33.0	1.19	37.7	1.03
DMSO Control	18.0	1.00	25.0	1.00	82.3	1.00	101.3	1.00	9.7	1.00	12.7	1.00	7.0	1.00	8.3	1.00	27.7	1.00	36.7	1.00
Ultrapure Water Control	–	–	–	–	81.7	1.00	–	–	8.7	1.00	–	–	–	–	–	–	32.0	1.00	–	–
1600	41.7	2.31	25.7	1.03	99.0	1.20	139.0	1.37	10.0	1.03	11.0	0.87	0.0	0.00	52.3	6.28	28.3	1.02	31.3	0.85
500	40.0	2.22	26.7	1.07	83.3	1.01	113.7	1.12	11.0	1.14	11.0	0.87	1.0	0.14	24.0	2.88	34.7	1.25	35.0	0.95
160	47.3	2.63	32.0	1.28	85.0	1.03	139.0	1.37	10.3	1.07	14.3	1.13	13.3	1.90	141.3	16.96	36.7	1.33	42.0	1.15
50	50.7	2.81	29.3	1.17	81.3	0.99	116.0	1.14	8.7	0.90	10.7	0.84	58.3	8.33	68.7	8.24	34.0	1.23	40.3	1.10
16	31.7	1.76	26.0	1.04	93.7	1.14	103.7	1.02	10.0	1.03	11.0	0.87	37.3	5.33	32.3	3.88	41.3	1.49	38.0	1.04
5	24.0	1.33	22.7	0.91	92.0	1.12	96.0	0.95	9.3	0.97	10.7	0.84	27.7	3.95	13.0	1.56	28.3	1.02	34.0	0.93
1.6	24.7	1.37	25.7	1.03	81.3	0.99	97.0	0.96	10.0	1.03	10.0	0.79	13.7	1.95	12.0	1.44	33.0	1.19	28.0	0.76
NPD (4mg)	199.3	11.07	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	944.0	11.56	–	–	1377.3	158.92	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	960.3	137.19	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	889.3	27.79	–	–
2AA (2mg)	–	–	2757.3	110.29	–	–	2526.7	24.93	–	–	193.7	15.29	–	–	311.7	37.40	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	181.0	4.94

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 solvent of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as solvent 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.

Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimurium
	TA 1537
	-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR
Untreated Control	9.7	1.53	11.3	1.00
DMSO Control	6.3	1.00	11.3	1.00
1600	10.7	1.68	21.0	1.85
1000	10.7	1.68	27.3	2.41
500	3.3	0.53	57.3	5.06
250	12.7	2.00	101.7	8.97
160	14.3	2.26	102.0	9.00
100	42.0	6.63	83.3	7.35
50	26.3	4.16	42.0	3.71
16	36.3	5.74	26.7	2.35
5	53.7	8.47	12.3	1.09
9AA (50mg)	622.7	98.32	–	–
2AA (2mg)	–	–	183.3	16.18

MR:Mutation Rate;9AA:9-Aminoacridine;2AA: 2-aminoanthracene

Remarks:DMSO was applied as solvent of the test item and positive control substances: 9AA and 2AA. The mutation rate of the test item, the untreated control, the 9AA and the 9AA is given referring to the DMSO.

Results of the Concentration Range Finding Test inSalmonella typhimuriumTA98

Test Item:		Leuco Sulfur Blue 20P
Date of Experiment:		May 23-25, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA98
Cell count (Overnight culture):		2.54 x 10^9CFU/mL
Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			19	14	16	16.3	‑	2.52	0.92
DMSO Control			16	18	19	17.7	‑	1.53	1.00
5000			18	19	24	20.3	P	3.21	1.15
1600			28	14	24	22.0	P	7.21	1.25
500			16	17	16	16.3	SP	0.58	0.92
160			13	12	14	13.0	SP	1.00	0.74
50			14	19	10	14.3	‑	4.51	0.81
16			15	18	27	20.0	‑	6.24	1.13
5			24	22	14	20.0	‑	5.29	1.13
Positive reference control (NPD)(4 µg/plate)			204	222	154	193.3	‑	35.23	10.94
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			17	24	14	18.3	‑	5.13	0.82
DMSO Control			15	22	30	22.3	‑	7.51	1.00
5000			12	19	10	13.7	P	4.73	0.61
1600			17	18	21	18.7	P	2.08	0.84
500			36	15	20	23.7	SP	10.97	1.06
160			26	33	21	26.7	‑	6.03	1.19
50			26	29	33	29.3	‑	3.51	1.31
16			32	43	35	36.7	‑	5.69	1.64
5			37	34	30	33.7	‑	3.51	1.51
Positive reference control (2AA)(2 µg/plate)			640	1360	1200	1066.7	‑	378.07	47.76

Obs : Observation (made by naked eye)                            P   : Precipitate

SD   : Standard Deviation                                                      SP :Slight precipitate

MR : Mutation Rate                                                              ‑    : Normal background lawn development, no precipitate

NPD:4-Nitro-1,2-phenylenediamine

2AA:2-aminoanthracene

 

Remark:        DMSO was applied as solvent of the test item and the positive control substances NPD and 2AA. The mutation rate of the test item, the untreated control the NPD and 2AA is given referring to the DMSO.

Results of the Concentration Range Finding Test inSalmonella typhimuriumTA100

Test Item:		Leuco Sulfur Blue 20P
Date of Experiment:		May 23-25, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA100
Cell count (Overnight culture):		2.41 x 10^9CFU/mL
Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			77	90	90	85.7	‑	7.51	0.94
DMSO Control			89	87	96	90.7	‑	4.73	1.00
Ultrapure Water Control			77	83	98	86.0	‑	10.82	1.00
5000			89	88	79	85.3	P	5.51	0.94
1600			89	85	105	93.0	P	10.58	1.03
500			117	101	112	110.0	SP	8.19	1.21
160			102	94	97	97.7	SP	4.04	1.08
50			106	84	100	96.7	‑	11.37	1.07
16			95	111	93	99.7	‑	9.87	1.10
5			96	86	96	92.7	‑	5.77	1.02
Positive reference control (SAZ)(2 µg/plate)			648	600	1080	776.0	‑	264.36	9.02
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			121	109	130	120.0	‑	10.54	1.10
DMSO Control			105	109	112	108.7	‑	3.51	1.00
5000			86	94	86	88.7	P	4.62	0.82
1600			100	90	92	94.0	P	5.29	0.87
500			108	108	100	105.3	SP	4.62	0.97
160			121	118	120	119.7	‑	1.53	1.10
50			110	107	122	113.0	‑	7.94	1.04
16			90	100	115	101.7	‑	12.58	0.94
5			96	106	110	104.0	‑	7.21	0.96
Positive reference control (2AA)(2 µg/plate)			2328	2008	1716	2017.3	‑	306.11	18.56

Obs : Observation (made by naked eye)                            P   : Precipitate

SD   : Standard Deviation                                                      SP :Slight precipitate

MR : Mutation Rate                                                              ‑    : Normal background lawn development, no precipitate

SAZ:Sodium azide

2AA:2-aminoanthracene

Remark:        DMSO was applied as solvent of the test item and the positive control substance 2AA. The ultrapure water was applied as solvent of the positive control substance SAZ. The mutation rate of the test item the untreated control and 2AA is given referring to the DMSO, the mutation rate of SAZ is given referring to ultrapure water.

 

 

Results of the Initial Mutation Test inSalmonella typhimuriumTA98

Test Item:		Leuco Sulfur Blue 20P
Date of Experiment:		July 19 – 21, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA98
Cell count (Overnight culture):		1.73 x 10^9CFU/mL
Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			13	20	19	17.3	‑	3.79	0.96
DMSO Control			16	18	20	18.0	‑	2.00	1.00
1600			41	38	46	41.7	P	4.04	2.31
500			41	34	45	40.0	P	5.57	2.22
160			37	53	52	47.3	‑	8.96	2.63
50			44	53	55	50.7	‑	5.86	2.81
16			37	27	31	31.7	‑	5.03	1.76
5			23	25	24	24.0	‑	1.00	1.33
1.6			29	29	16	24.7	‑	7.51	1.37
Positive reference control (NPD)(4 µg/plate)			206	170	222	199.3	‑	26.63	11.07
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			22	25	23	23.3	‑	1.53	0.93
DMSO Control			24	26	25	25.0	‑	1.00	1.00
1600			27	24	26	25.7	P	1.53	1.03
500			37	18	25	26.7	P	9.61	1.07
160			39	26	31	32.0	‑	6.56	1.28
50			31	26	31	29.3	‑	2.89	1.17
16			22	28	28	26.0	‑	3.46	1.04
5			21	29	18	22.7	‑	5.69	0.91
1.6			28	25	24	25.7	‑	2.08	1.03
Positive reference control (2AA)(2 µg/plate)			3024	2664	2584	2757.3	‑	234.38	110.29

Obs : Observation (made by naked eye)                            P   : Precipitate

SD   : Standard Deviation                                                      ‑    : Normal background lawn development, no precipitate

MR : Mutation Rate                                                              

NPD:4-Nitro-1,2-phenylenediamine                                 

2AA:2-aminoanthracene

 

Remark:        DMSO was applied as solvent of the test item and the positive control substances NPD and 2AA. The mutation rate of the test item, the untreated control the NPD and 2AA is given referring to the DMSO.

Results of the Initial Mutation Test inSalmonella typhimuriumTA100

Test Item:		Leuco Sulfur Blue 20P
Date of Experiment:		July 19 – 21, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA100
Cell count (Overnight culture):		1.69 x 10^9CFU/mL
Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			98	86	82	88.7	‑	8.33	1.08
DMSO Control			83	81	83	82.3	‑	1.15	1.00
Ultrapure Water Control			82	84	79	81.7	‑	2.52	1.00
1600			100	116	81	99.0	P	17.52	1.20
500			96	82	72	83.3	P	12.06	1.01
160			91	83	81	85.0	‑	5.29	1.03
50			85	88	71	81.3	‑	9.07	0.99
16			104	90	87	93.7	‑	9.07	1.14
5			97	84	95	92.0	‑	7.00	1.12
1.6			81	92	71	81.3	‑	10.50	0.99
Positive reference control (SAZ)(2 µg/plate)			872	1016	944	944.0	‑	72.00	11.56
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			105	96	131	110.7	‑	18.18	1.09
DMSO Control			94	95	115	101.3	‑	11.85	1.00
1600			114	152	151	139.0	P	21.66	1.37
500			115	103	123	113.7	P	10.07	1.12
160			149	140	128	139.0	‑	10.54	1.37
50			111	111	126	116.0	‑	8.66	1.14
16			107	101	103	103.7	‑	3.06	1.02
5			106	92	90	96.0	‑	8.72	0.95
1.6			99	94	98	97.0	‑	2.65	0.96
Positive reference control (2AA)(2 µg/plate)			2600	2512	2468	2526.7	‑	67.21	24.93

Obs : Observation (made by naked eye)                            P   : Precipitate

SD   : Standard Deviation                                                      ‑    : Normal background lawn development, no precipitate

MR : Mutation Rate                                                              

SAZ:Sodium azide

2AA:2-aminoanthracene

 

Remark:        DMSO was applied as solvent of the test item and the positive control substance 2AA. The ultrapure water was applied as solvent of the positive control substance SAZ. The mutation rate of the test item the untreated control and 2AA is given referring to the DMSO, the mutation rate of SAZ is given referring to ultrapure water.

Results of the Initial Mutation Test inSalmonella typhimuriumTA1535

Test Item:		Leuco Sulfur Blue 20P
Date of Experiment:		July 19 – 21, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA1535
Cell count (Overnight culture):		2.09 x 10^9CFU/mL
Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			6	10	13	9.7	‑	3.51	1.00
DMSO Control			9	11	9	9.7	‑	1.15	1.00
Ultrapure Water Control			8	8	10	8.7	‑	1.15	1.00
1600			7	8	15	10.0	P	4.36	1.03
500			12	11	10	11.0	P	1.00	1.14
160			10	14	7	10.3	‑	3.51	1.07
50			6	13	7	8.7	‑	3.79	0.90
16			12	8	10	10.0	‑	2.00	1.03
5			10	11	7	9.3	‑	2.08	0.97
1.6			11	8	11	10.0	‑	1.73	1.03
Positive reference control (SAZ)(2 µg/plate)			1320	1392	1420	1377.3	‑	51.59	158.92
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			9	14	14	12.3	‑	2.89	0.97
DMSO Control			19	10	9	12.7	‑	5.51	1.00
1600			8	11	14	11.0	P	3.00	0.87
500			14	9	10	11.0	P	2.65	0.87
160			14	18	11	14.3	‑	3.51	1.13
50			15	10	7	10.7	‑	4.04	0.84
16			15	8	10	11.0	‑	3.61	0.87
5			11	12	9	10.7	‑	1.53	0.84
1.6			12	6	12	10.0	‑	3.46	0.79
Positive reference control (2AA)(2 µg/plate)			118	242	221	193.7	‑	66.37	15.29

Obs : Observation (made by naked eye)                            P   : Precipitate

SD   : Standard Deviation                                                      ‑    : Normal background lawn development, no precipitate

MR : Mutation Rate                                                              

SAZ:Sodium azide

2AA:2-aminoanthracene

 

Remark:        DMSO was applied as solvent of the test item and the positive control substance 2AA. The ultrapure water was applied as solvent of the positive control substance SAZ. The mutation rate of the test item the untreated control and 2AA is given referring to the DMSO, the mutation rate of SAZ is given referring to ultrapure water.

Results of the Initial Mutation Test inSalmonella typhimurium TA1537

Test Item:		Leuco Sulfur Blue 20P
Date of Experiment:		July 19 – 21, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA1537
Cell count (Overnight culture):		1.24 x 10^9CFU/mL
Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			5	6	8	6.3	‑	1.53	0.90
DMSO Control			7	6	8	7.0	‑	1.00	1.00
1600			0	0	0	0.0	B, P	0.00	0.00
500			0	2	1	1.0	B, P	1.00	0.14
160			6	20	14	13.3	SB	7.02	1.90
50			55	55	65	58.3	‑	5.77	8.33
16			40	32	40	37.3	‑	4.62	5.33
5			29	33	21	27.7	‑	6.11	3.95
1.6			16	8	17	13.7	‑	4.93	1.95
Positive reference control (9AA)(50 µg/plate)			847	758	1276	960.3	‑	276.97	137.19
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			4	11	6	7.0	‑	3.61	0.84
DMSO Control			8	10	7	8.3	‑	1.53	1.00
1600			55	59	43	52.3	SB, P	8.33	6.28
500			24	24	24	24.0	SB, P	0.00	2.88
160			134	160	130	141.3	‑	16.29	16.96
50			64	70	72	68.7	‑	4.16	8.24
16			29	31	37	32.3	‑	4.16	3.88
5			15	12	12	13.0	‑	1.73	1.56
1.6			16	10	10	12.0	‑	3.46	1.44
Positive reference control (2AA)(2 µg/plate)			287	323	325	311.7	‑	21.39	37.40

Obs : Observation (made by naked eye)                            P   : Precipitate

SD   : Standard Deviation                                                      B   :Reduced background lawn development

MR : Mutation Rate                                                              SB : Slightly reduced background lawn development

9AA:9-Aminoacridine                                                          ‑    : Normal background lawn development, no precipitate

2AA:2-aminoanthracene

 

Remark:        DMSO was applied as solvent of the test item and the positive control substances 9AA and 2AA. The mutation rate of the test item, the untreated control the 9AA and 2AA is given referring to the DMSO.

Results of the Initial Mutation Test inEscherichia coliWP2uvrA

Test Item:		Leuco Sulfur Blue 20P
Date of Experiment:		July 19 – 21, 2017
Applied Method:		Plate Incorporation
Strain:		Escherichia coliWP2uvrA
Cell count (Overnight culture):		3.67 x 10^9CFU/mL
Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			42	29	28	33.0	‑	7.81	1.19
DMSO Control			20	33	30	27.7	‑	6.81	1.00
Ultrapure Water Control			32	30	34	32.0	‑	2.00	1.00
1600			33	25	27	28.3	P	4.16	1.02
500			33	34	37	34.7	P	2.08	1.25
160			35	29	46	36.7	‑	8.62	1.33
50			28	34	40	34.0	‑	6.00	1.23
16			35	47	42	41.3	‑	6.03	1.49
5			23	29	33	28.3	‑	5.03	1.02
1.6			28	41	30	33.0	‑	7.00	1.19
Positive reference control (MMS)(2 µL/plate)			988	888	792	889.3	‑	98.01	27.79
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			40	37	36	37.7	‑	2.08	1.03
DMSO Control			36	30	44	36.7	‑	7.02	1.00
1600			33	30	31	31.3	P	1.53	0.85
500			41	37	27	35.0	P	7.21	0.95
160			44	38	44	42.0	‑	3.46	1.15
50			47	41	33	40.3	‑	7.02	1.10
16			36	38	40	38.0	‑	2.00	1.04
5			32	34	36	34.0	‑	2.00	0.93
1.6			22	34	28	28.0	‑	6.00	0.76
Positive reference control (2AA)(50 µg/plate)			184	169	190	181.0	‑	10.82	4.94

Obs : Observation (made by naked eye)                            P   : Precipitate

SD   : Standard Deviation                                                      ‑    : Normal background lawn development, no precipitate

MR : Mutation Rate                                                              

MMS:Methyl methanesulfonate

2AA:2-aminoanthracene

 

Remark:        DMSO was applied as solvent of the test item and the positive control substance 2AA. The ultrapure water was applied as solvent of the positive control substance MMS. The mutation rate of the test item the untreated control and 2AA is given referring to the DMSO, the mutation rate of MMS is given referring to ultrapure water.

 

Results of the Confirmatory Mutation Test inSalmonella typhimuriumTA1537

Test Item:		Leuco Sulfur Blue 20P
Date of Experiment:		October 18 – 20, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA1537
Cell count (Overnight culture):		1.67 x 10^9CFU/mL
Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			14	6	9	9.7	‑	4.04	1.53
DMSO Control			4	11	4	6.3	‑	4.04	1.00
1600			11	6	15	10.7	B, P	4.51	1.68
1000			6	13	13	10.7	B, P	4.04	1.68
500			0	10	0	3.3	B, SP	5.77	0.53
250			10	15	13	12.7	B	2.52	2.00
160			18	11	14	14.3	SB	3.51	2.26
100			34	44	48	42.0	‑	7.21	6.63
50			30	27	22	26.3	‑	4.04	4.16
16			39	33	37	36.3	‑	3.06	5.74
5			51	56	54	53.7	‑	2.52	8.47
Positive reference control (9AA)(50 µg/plate)			512	636	720	622.7	‑	104.64	98.32
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			13	13	8	11.3	‑	2.89	1.00
DMSO Control			8	17	9	11.3	‑	4.93	1.00
1600			19	28	16	21.0	SB, P	6.24	1.85
1000			24	29	29	27.3	SB, P	2.89	2.41
500			68	42	62	57.3	SB, P	13.61	5.06
250			98	111	96	101.7	SP	8.14	8.97
160			96	104	106	102.0	‑	5.29	9.00
100			105	55	90	83.3	‑	25.66	7.35
50			38	47	41	42.0	‑	4.58	3.71
16			24	24	32	26.7	‑	4.62	2.35
5			14	12	11	12.3	‑	1.53	1.09
Positive reference control (2AA)(2 µg/plate)			202	164	184	183.3	‑	19.01	16.18

Obs : Observation (made by naked eye)                            P      :Precipitate

SD   : Standard Deviation                                                      SP   :Slight precipitate

MR : Mutation Rate                                                              B      :Reduced background lawn development

9AA:9-Aminoacridine                                                          SB    :Slightly reduced background lawn development

2AA:2-aminoanthracene                                                        ‑    : Normal background lawn development, no precipitate

 

Remark:        DMSO was applied as solvent of the test item and the positive control substances 9AA and 2AA. The mutation rate of the test item, the untreated control the 9AA and 2AA is given referring to the DMSO.

 

Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.0	105.0	10.5	8.1	25.4
		SD	3.7	25.7	1.4	2.3	5.2
		Minimum	9	66	3	2	11
		Maximum	39	155	23	19	45
		n	226	236	216	214	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.5	117.1	11.8	9.0	33.9
		SD	4.3	18.1	1.4	1.9	5.2
		Minimum	12	75	4	2	17
		Maximum	46	166	23	20	56
		n	226	236	216	214	215
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	20.4	100.1	10.3	7.9	24.7
		SD	3.6	24.8	1.3	2.4	4.6
		Minimum	10	64	3	2	11
		Maximum	38	147	23	20	45
		n	226	236	216	214	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	26.5	113.8	11.8	8.8	33.7
		SD	4.1	18.3	1.5	1.9	5.0
		Minimum	15	71	3	3	16
		Maximum	47	162	25	20	57
		n	226	236	216	214	215
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.9	104.7	10.5	7.6	26.1
		SD	3.7	25.9	1.5	2.2	5.5
		Minimum	12	68	3	2	12
		Maximum	35	154	24	16	48
		n	89	236	216	89	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.4	117.3	11.4	8.7	34.9
		SD	4.0	18.5	1.3	2.2	4.9
		Minimum	15	83	4	3	18
		Maximum	43	167	22	16	57
		n	89	152	149	89	148

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

                                               SD: Standard deviation;    DMSO: Dimethyl sulfoxide; n: number of studies

Historical Control Values for Revertants/Plate (for the Period of 2008-2016) (continued)

			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	260.1	977.2	847.3	478.6	724.5
		SD	31.8	150.6	126.3	104.5	65.0
		Minimum	123	521	359	110	320
		Maximum	664	1970	1855	1601	1313
		n	226	236	216	214	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1222.7	1436.4	164.1	147.0	257.7
		SD	274.9	318.3	33.1	20.1	72.5
		Minimum	386	583	85	69	140
		Maximum	2676	2988	498	399	477
		n	226	236	216	214	215

Abbreviations:   TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535,

                               TA1537;E. coli:Escherichia coliWP2uvrA

                                     SD: Standard deviation;   DMSO: Dimethyl sulfoxide;  n: number of studies

 

Applicant's summary and conclusion

Conclusions:

The test item showed a mutagenic activity on Salmonella typhimurium TA1537 carrying frameshift mutation in the absence and presence of exogenous metabolic activation, under the test conditions used in this study.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The study included preliminary solubility tests, preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (repeated plate incorporation test). The initial mutation test was 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 WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. The confirmatory mutation test was carried out using Salmonella typhimurium TA1537. Based on the results of the solubility test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content (92.5 %) was made in the experiments. 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 mutation test: ±S9 mix:1600; 500; 160; 50; 16; 5 and 1.6 µg/plate. The selection of the concentration range was based on the recommendations in OECD 471 guideline. At the concentration choice the non-toxicity of the test item and the appearance of precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye. To confirm and to investigate the reproducibility of the positive result of the initial mutation test the following nine concentration levels were investigated in the confirmatory mutation test: ±S9 mix: 1600, 1000, 500, 250, 160, 100, 50, 16 and 5 µg/plate.

When evaluated by the naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains down to and including the concentration level of 500 µg/plate in the absence and down to and including the concentration level of 250 µg/plate in the presence of exogenous metabolic activation following the plate incorporation procedures. In the initial mutation test an inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain in the absence (in the concentration range of 1600-160 µg/plate) and also in the presence (at the concentrations of 1600 and 500 µg/plate) of exogenous metabolic activation. The confirmatory mutation test repeated the initial mutation test results. In both experimental phases the inhibitory effect was indicated by absent or decreased revertant colony counts (compared to the revertant colony numbers of the DMSO control) and/or 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 solvent control (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 biologically relevant increases (more than 3-fold increase)in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges (or even were above the historical control data range), thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. Unequivocal positive results, confirmed by a repeat of the experiment, were noticed following the plate incorporation procedures (initial and confirmatory mutation tests) in the investigated Salmonella typhimurium TA1537 strain (±S9 Mix). The obtained revertant colony number increases were clearly above the corresponding historical control data range and the relevant genotoxicological threshold for being positive in both experiments at the concentration range of 50‑5 µg/plate (-S9 mix) and at the concentrations range of 160-50 µg/plate (+S9 mix). The obtained increased tendencies followed clear dose-relationship. Under the experimental conditions applied, the test item induced gene mutations by frameshifts in the genome of the Salmonella typhimurium TA1537 tester strain examined. In conclusion, the test item showed a mutagenic activity on Salmonella typhimurium TA1537 carrying frameshift mutation in the absence and presence of exogenous metabolic activation, under the test conditions used in this study.