Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

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:

20 October 2017 - 10 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

EC Number:

291-833-0

EC Name:

Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

Cas Number:

90480-94-7

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-aminophenol with 4-​(2-​naphthalenylamino)​phenol and sodium polysulfide

Test material form:

solid

Details on test material:

Test item: Leuco Sulphur Black 11
Appearance: black solid
CAS No: 90480-94-7
EC No: 291-833-0

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:

5000; 1600; 500; 160; 50 and 16 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:

Origin of the Bacterial Strains

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 10-13 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:

Justification of concentrations:
Selection of the concentration range for the initial mutation test was done on the basis of the referred OECD Guideline 471, on the basis of the solubility test
and concentration range finding test (informatory toxicity 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-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 same concentration levels were investigated
in
the confirmatory mutation test:
±S9 mix: 5000; 1600; 500; 160; 50 and 16 µg/plate.

The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98,
TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
In the informatory toxicity test the revertant colony numbers of the solvent control plates with and without S9 mix were in line with the corresponding
historical control data ranges. The positive control treatments showed the expected biological relevant increases in induced revertant colonies in both tester
strains.

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 1535

Metabolic activation:

with and without

Genotoxicity:

positive

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 100

Metabolic activation:

with and without

Genotoxicity:

positive

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 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 4

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 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 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 concentration range finding test and six concentration levels were investigated in the initial mutation test and 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. 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 the Salmonella typhimurium TA98, TA1537 and Escherichia coli WP2 uvrA strains in the absence and also in the presence of an exogenous metabolic activation system (±S9 mix).Unequivocal positive results were noticed following treatment with the test item in the investigated Salmonella typhimurium TA100 and TA1535 strains (±S9 mix). The obtained revertant colony number increases were clearly above the corresponding historical control data ranges and the relevant genotoxicological threshold for being positive in TA100 at the concentrations of 5000 and 1600 µg/plate (±S9 mix); in TA1535 in the concentration range of 5000-500 µg/plate (±S9 mix). The obtained increased tendencies followed clear dose-relationship. Furthermore significantly increased revertant colony numbers (above the corresponding historical control data ranges; however below the genotoxicological threshold for being positive) were noticed in Escherichia coli WP2 uvrA (±S9 mix). The increased revertant colony numbers were above the corresponding historical control data range; however below the genotoxicological threshold for being positive in S. typhimurium TA1535 at 160 µg/plate (-S9 mix), in E. coli WP2 uvrA 5000, 1600 and 500 µg/plate (-S9 mix), and at 5000 and 1600 µg/plate (+S9 mix); furthermore in S. typhimurium TA1537 at 5000 µg/plate (+S9 mix). The obtained increased tendencies in E. coli WP2 uvrA followed clear dose-relationship. No substantial increases were observed in revertant colony numbers of S. typhimurium TA98 (±S9 mix) and (with exception of the unique increase at 5000 µg/plate (+S9 mix)) S. typhimurium TA1537 (±S9 mix). In this test inhibitory, cytotoxic effects of the test item were not observed. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) were considered to be within the biological variability range of the applied test system. When evaluated by naked eye, non-interfering test item precipitate (test item particles) was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 µg/plate in the absence and presence of an exogenous metabolic activation (±S9 mix).

To confirm and to investigate the reproducibility of this positive result a confirmatory mutation test was performed with S. typhimurium TA100 and TA1535 strains in the absence and presence of exogenous metabolic activation (±S9 mix). The strains Salmonella typhimurium TA98, TA1537 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 TA100 and TA1535 strains (±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 in strain TA100 at the highest examined concentration of 5000 µg/plate (±S9 mix); and in strain TA1535 in the concentration range of 5000-500 µg/plate (±S9 mix). The obtained increased tendencies, especially in the latter case, followed a clear dose-relationship.The increased revertant colony numbers remained below the corresponding historical control data range and below the threshold for being positive; however followed a dose-relationship in Salmonella typhimurium TA100, at 1600 µg/plate (±S9 mix) and in TA1535 at 160 and 50 µg/plate (±S9 mix). Similarly to the initial mutation test results, in the confirmatory mutation test an inhibitory, cytotoxic effect of the test item was not observed. When evaluated by naked eye, non-interfering test item precipitate (test item particles) was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 µg/plate in the absence and presence of an exogenous metabolic activation (±S9 mix).

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 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	20.7	1.00	26.3	1.05	86.3	1.10	105.0	1.16
DMSO Control	20.7	1.00	25.0	1.00	78.7	1.00	90.3	1.00
Ultrapure Water Control	–	–	–	–	84.7	1.00	–	–
5000	21.3	1.03	16.7	0.67	120.3	1.53	123.3	1.37
1600	16.3	0.79	15.0	0.60	82.7	1.05	92.0	1.02
500	17.7	0.85	16.0	0.64	77.3	0.98	85.0	0.94
160	20.3	0.98	22.7	0.91	67.7	0.86	82.3	0.91
50	10.0	0.48	25.7	1.03	70.7	0.90	77.0	0.85
16	18.7	0.90	22.3	0.89	82.0	1.04	78.0	0.86
5	19.0	0.92	19.7	0.79	77.3	0.98	98.0	1.08
NPD (4mg)	223.0	10.79	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	949.3	11.21	–	–
2AA (2mg)	–	–	1421.3	56.85	–	–	2406.7	26.64

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	29.0	1.67	28.0	0.97	92.3	1.26	88.7	1.09	10.0	0.79	11.3	1.21	12.3	1.16	9.7	0.88	29.7	0.96	42.0	0.85
DMSO Control	17.3	1.00	29.0	1.00	73.0	1.00	81.0	1.00	12.7	1.00	9.3	1.00	10.7	1.00	11.0	1.00	31.0	1.00	49.3	1.00
Ultrapure Water Control	–	–	–	–	81.0	1.00	–	–	9.7	1.00	–	–	–	–	–	–	45.7	1.00	–	–
5000	17.3	1.00	25.7	0.89	207.3	2.84	252.3	3.12	297.3	23.47	213.7	22.89	13.7	1.28	20.7	1.88	84.0	2.71	76.7	1.55
1600	21.3	1.23	31.0	1.07	147.7	2.02	189.0	2.33	163.0	12.87	128.3	13.75	14.7	1.38	8.0	0.73	74.3	2.40	65.3	1.32
500	23.7	1.37	28.0	0.97	115.7	1.58	123.3	1.52	62.3	4.92	52.3	5.61	13.0	1.22	11.3	1.03	50.0	1.61	43.3	0.88
160	21.7	1.25	32.7	1.13	107.3	1.47	110.3	1.36	33.3	2.63	18.7	2.00	14.3	1.34	13.7	1.24	39.7	1.28	37.3	0.76
50	19.0	1.10	31.3	1.08	100.7	1.38	94.0	1.16	14.0	1.11	13.0	1.39	11.0	1.03	11.3	1.03	41.3	1.33	40.0	0.81
16	25.0	1.44	32.3	1.11	104.3	1.43	108.0	1.33	17.3	1.37	11.7	1.25	10.3	0.97	11.7	1.06	30.0	0.97	47.7	0.97
NPD (4mg)	371.3	21.42	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1058.7	13.07	–	–	1088.7	112.62	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	925.3	86.75	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	575.3	12.60	–	–
2AA (2mg)	–	–	1718.7	59.26	–	–	1952.0	24.10	–	–	202.0	21.64	–	–	188.0	17.09	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	244.7	4.96

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 typhimuriumtester strains
	TA 100				TA 1535
	-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	96.3	1.08	101.3	0.97	8.7	0.84	12.7	1.19
DMSO Control	89.0	1.00	104.3	1.00	10.3	1.00	10.7	1.00
Ultrapure Water Control	92.7	1.00	–	–	9.0	1.00	–	–
5000	224.7	2.52	261.0	2.50	185.3	17.94	248.3	23.28
1600	140.0	1.57	161.3	1.55	84.7	8.19	100.0	9.38
500	96.0	1.08	110.0	1.05	41.7	4.03	46.0	4.31
160	86.0	0.97	95.3	0.91	15.3	1.48	20.0	1.88
50	84.0	0.94	94.3	0.90	13.7	1.32	17.3	1.63
16	97.0	1.09	91.3	0.88	11.0	1.06	11.7	1.09
SAZ (2mg)	1173.3	12.66	–	–	880.0	97.78	–	–
2AA (2mg)	–	–	1834.7	17.58	–	–	256.7	24.06

MR:Mutation Rate;SAZ: Sodium azide;2AA: 2-aminoanthracene

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

Results of the Concentration Range Finding Test inSalmonella typhimuriumTA98

Test Item:		Leuco Sulphur Black 11
Date of Experiment:		July 26-28, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA98
Cell count (Overnight culture):		1.10 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			21	20	21	20.7	‑	0.58	1.00
DMSO Control			19	23	20	20.7	‑	2.08	1.00
5000			23	19	22	21.3	P	2.08	1.03
1600			13	21	15	16.3	SP	4.16	0.79
500			17	18	18	17.7	‑	0.58	0.85
160			18	24	19	20.3	‑	3.21	0.98
50			10	10	10	10.0	‑	0.00	0.48
16			21	17	18	18.7	‑	2.08	0.90
5			21	14	22	19.0	‑	4.36	0.92
Positive reference control (NPD)(4 µg/plate)			245	203	221	223.0	‑	21.07	10.79
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			24	28	27	26.3	‑	2.08	1.05
DMSO Control			23	20	32	25.0	‑	6.24	1.00
5000			16	14	20	16.7	P	3.06	0.67
1600			17	12	16	15.0	SP	2.65	0.60
500			15	15	18	16.0	‑	1.73	0.64
160			23	21	24	22.7	‑	1.53	0.91
50			31	24	22	25.7	‑	4.73	1.03
16			22	22	23	22.3	‑	0.58	0.89
5			19	22	18	19.7	‑	2.08	0.79
Positive reference control (2AA)(2 µg/plate)			1712	1416	1136	1421.3	‑	288.04	56.85

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 Sulphur Black 11
Date of Experiment:		July 26-28, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA100
Cell count (Overnight culture):		1.47 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			87	82	90	86.3	‑	4.04	1.10
DMSO Control			84	69	83	78.7	‑	8.39	1.00
Ultrapure Water Control			87	80	87	84.7	‑	4.04	1.00
5000			103	130	128	120.3	P	15.04	1.53
1600			77	83	88	82.7	SP	5.51	1.05
500			73	76	83	77.3	‑	5.13	0.98
160			63	66	74	67.7	‑	5.69	0.86
50			74	84	54	70.7	‑	15.28	0.90
16			77	73	96	82.0	‑	12.29	1.04
5			90	73	69	77.3	‑	11.15	0.98
Positive reference control (SAZ)(2 µg/plate)			928	976	944	949.3	‑	24.44	11.21
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			95	113	107	105.0	‑	9.17	1.16
DMSO Control			95	87	89	90.3	‑	4.16	1.00
5000			112	147	111	123.3	P	20.50	1.37
1600			91	92	93	92.0	SP	1.00	1.02
500			74	93	88	85.0	‑	9.85	0.94
160			86	85	76	82.3	‑	5.51	0.91
50			84	81	66	77.0	‑	9.64	0.85
16			87	77	70	78.0	‑	8.54	0.86
5			112	94	88	98.0	‑	12.49	1.08
Positive reference control (2AA)(2 µg/plate)			2480	2410	2330	2406.7	‑	75.06	26.64

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 Sulphur Black 11
Date of Experiment:		October 24 – 26, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA98
Cell count (Overnight culture):		1.84 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			27	30	30	29.0	‑	1.73	1.67
DMSO Control			15	18	19	17.3	‑	2.08	1.00
5000			20	15	17	17.3	TIP	2.52	1.00
1600			22	15	27	21.3	TIP	6.03	1.23
500			25	22	24	23.7	‑	1.53	1.37
160			20	25	20	21.7	‑	2.89	1.25
50			17	22	18	19.0	‑	2.65	1.10
16			19	18	38	25.0	‑	11.27	1.44
Positive reference control (NPD)(4 µg/plate)			412	382	320	371.3	‑	46.92	21.42
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			30	31	23	28.0	‑	4.36	0.97
DMSO Control			29	31	27	29.0	‑	2.00	1.00
5000			24	29	24	25.7	TIP	2.89	0.89
1600			32	32	29	31.0	TIP	1.73	1.07
500			34	23	27	28.0	‑	5.57	0.97
160			31	31	36	32.7	‑	2.89	1.13
50			24	33	37	31.3	‑	6.66	1.08
16			26	36	35	32.3	‑	5.51	1.11
Positive reference control (2AA)(2 µg/plate)			1704	1828	1624	1718.7	‑	102.79	59.26

Obs : Observation (made by naked eye)                     TIP  : Test Item Particles

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 Sulphur Black 11
Date of Experiment:		October 24 – 26, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA100
Cell count (Overnight culture):		1.40 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			94	89	94	92.3	‑	2.89	1.26
DMSO Control			70	73	76	73.0	‑	3.00	1.00
Ultrapure Water Control			73	80	90	81.0	‑	8.54	1.00
5000			197	205	220	207.3	TIP	11.68	2.84
1600			155	146	142	147.7	TIP	6.66	2.02
500			110	107	130	115.7	‑	12.50	1.58
160			101	103	118	107.3	‑	9.29	1.47
50			104	96	102	100.7	‑	4.16	1.38
16			109	106	98	104.3	‑	5.69	1.43
Positive reference control (SAZ)(2 µg/plate)			1040	1080	1056	1058.7	‑	20.13	13.07
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			82	98	86	88.7	‑	8.33	1.09
DMSO Control			77	79	87	81.0	‑	5.29	1.00
5000			292	236	229	252.3	TIP	34.53	3.12
1600			201	175	191	189.0	TIP	13.11	2.33
500			122	113	135	123.3	‑	11.06	1.52
160			118	113	100	110.3	‑	9.29	1.36
50			99	95	88	94.0	‑	5.57	1.16
16			107	117	100	108.0	‑	8.54	1.33
Positive reference control (2AA)(2 µg/plate)			2000	2016	1840	1952.0	‑	97.32	24.10

Obs : Observation (made by naked eye)                     TIP  : Test Item Particles

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 Sulphur Black 11
Date of Experiment:		October 24 – 26, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA1535
Cell count (Overnight culture):		2.15 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			15	7	8	10.0	‑	4.36	0.79
DMSO Control			12	10	16	12.7	‑	3.06	1.00
Ultrapure Water Control			10	14	5	9.7	‑	4.51	1.00
5000			312	284	296	297.3	TIP	14.05	23.47
1600			145	172	172	163.0	TIP	15.59	12.87
500			57	70	60	62.3	‑	6.81	4.92
160			28	32	40	33.3	‑	6.11	2.63
50			15	15	12	14.0	‑	1.73	1.11
16			18	19	15	17.3	‑	2.08	1.37
Positive reference control (SAZ)(2 µg/plate)			1136	1000	1130	1088.7	‑	76.85	112.62
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			13	9	12	11.3	‑	2.08	1.21
DMSO Control			11	13	4	9.3	‑	4.73	1.00
5000			229	198	214	213.7	TIP	15.50	22.89
1600			144	109	132	128.3	TIP	17.79	13.75
500			48	46	63	52.3	‑	9.29	5.61
160			17	13	26	18.7	‑	6.66	2.00
50			12	12	15	13.0	‑	1.73	1.39
16			12	11	12	11.7	‑	0.58	1.25
Positive reference control (2AA)(2 µg/plate)			204	182	220	202.0	‑	19.08	21.64

Obs : Observation (made by naked eye)                     TIP  : Test Item Particles

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 typhimuriumTA1537

Test Item:		Leuco Sulphur Black 11
Date of Experiment:		October 24 – 26, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA1537
Cell count (Overnight culture):		1.97 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			18	8	11	12.3	‑	5.13	1.16
DMSO Control			16	8	8	10.7	‑	4.62	1.00
5000			16	8	17	13.7	TIP	4.93	1.28
1600			14	12	18	14.7	TIP	3.06	1.38
500			14	13	12	13.0	‑	1.00	1.22
160			15	8	20	14.3	‑	6.03	1.34
50			7	12	14	11.0	‑	3.61	1.03
16			12	7	12	10.3	‑	2.89	0.97
Positive reference control (9AA)(50 µg/plate)			920	968	888	925.3	‑	40.27	86.75
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			14	7	8	9.7	‑	3.79	0.88
DMSO Control			15	11	7	11.0	‑	4.00	1.00
5000			22	18	22	20.7	TIP	2.31	1.88
1600			11	7	6	8.0	TIP	2.65	0.73
500			11	9	14	11.3	‑	2.52	1.03
160			15	10	16	13.7	‑	3.21	1.24
50			9	15	10	11.3	‑	3.21	1.03
16			10	12	13	11.7	‑	1.53	1.06
Positive reference control (2AA)(2 µg/plate)			192	171	201	188.0	‑	15.39	17.09

Obs : Observation (made by naked eye)                     TIP  : Test Item Particles

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

MR : Mutation Rate                                                              

9AA:9-Aminoacridine                                                          

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 Sulphur Black 11
Date of Experiment:		October 24 – 26, 2017
Applied Method:		Plate Incorporation
Strain:		Escherichia coliWP2uvrA
Cell count (Overnight culture):		3.23 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			27	36	26	29.7	‑	5.51	0.96
DMSO Control			27	37	29	31.0	‑	5.29	1.00
Ultrapure Water Control			46	49	42	45.7	‑	3.51	1.00
5000			87	80	85	84.0	TIP	3.61	2.71
1600			73	70	80	74.3	TIP	5.13	2.40
500			45	48	57	50.0	‑	6.24	1.61
160			39	39	41	39.7	‑	1.15	1.28
50			36	49	39	41.3	‑	6.81	1.33
16			31	33	26	30.0	‑	3.61	0.97
Positive reference control (MMS)(2 µL/plate)			645	549	532	575.3	‑	60.93	12.60
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			44	35	47	42.0	‑	6.24	0.85
DMSO Control			45	50	53	49.3	‑	4.04	1.00
5000			83	74	73	76.7	TIP	5.51	1.55
1600			76	63	57	65.3	TIP	9.71	1.32
500			46	46	38	43.3	‑	4.62	0.88
160			40	33	39	37.3	‑	3.79	0.76
50			32	48	40	40.0	‑	8.00	0.81
16			50	47	46	47.7	‑	2.08	0.97
Positive reference control (2AA)(50 µg/plate)			255	238	241	244.7	‑	9.07	4.96

Obs : Observation (made by naked eye)                     TIP  : Test Item Particles

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 typhimuriumTA100

Test Item:		Leuco Sulphur Black 11
Date of Experiment:		November 15 – 17, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA100
Cell count (Overnight culture):		1.32 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			97	99	93	96.3	‑	3.06	1.08
DMSO Control			87	90	90	89.0	‑	1.73	1.00
Ultrapure Water Control			101	86	91	92.7	‑	7.64	1.00
5000			228	236	210	224.7	TIP	13.32	2.52
1600			128	136	156	140.0	TIP	14.42	1.57
500			94	94	100	96.0	‑	3.46	1.08
160			72	94	92	86.0	‑	12.17	0.97
50			84	82	86	84.0	‑	2.00	0.94
16			87	106	98	97.0	‑	9.54	1.09
Positive reference control (SAZ)(2 µg/plate)			1192	1160	1168	1173.3	‑	16.65	12.66
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			116	109	79	101.3	‑	19.66	0.97
DMSO Control			104	109	100	104.3	‑	4.51	1.00
5000			222	261	300	261.0	TIP	39.00	2.50
1600			170	146	168	161.3	TIP	13.32	1.55
500			112	105	113	110.0	‑	4.36	1.05
160			88	104	94	95.3	‑	8.08	0.91
50			90	106	87	94.3	‑	10.21	0.90
16			94	82	98	91.3	‑	8.33	0.88
Positive reference control (2AA)(2 µg/plate)			1624	2128	1752	1834.7	‑	261.97	17.58

Obs : Observation (made by naked eye)                     TIP  : Test Item Particles

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 Confirmatory Mutation Test inSalmonella typhimuriumTA1535

Test Item:		Leuco Sulphur Black 11
Date of Experiment:		November 15 – 17, 2017
Applied Method:		Plate Incorporation
Strain:		Salmonella typhimuriumTA1535
Cell count (Overnight culture):		1.40 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			8	12	6	8.7	‑	3.06	0.84
DMSO Control			12	8	11	10.3	‑	2.08	1.00
Ultrapure Water Control			13	7	7	9.0	‑	3.46	1.00
5000			167	182	207	185.3	TIP	20.21	17.94
1600			75	89	90	84.7	TIP	8.39	8.19
500			36	53	36	41.7	‑	9.81	4.03
160			18	19	9	15.3	‑	5.51	1.48
50			14	14	13	13.7	‑	0.58	1.32
16			4	19	10	11.0	‑	7.55	1.06
Positive reference control (SAZ)(2 µg/plate)			824	880	936	880.0	‑	56.00	97.78
With Exogenous Metabolic Activation (+S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
	Parallel:		1	2	3
Untreated Control			10	13	15	12.7	‑	2.52	1.19
DMSO Control			8	14	10	10.7	‑	3.06	1.00
5000			239	252	254	248.3	TIP	8.14	23.28
1600			89	109	102	100.0	TIP	10.15	9.38
500			42	54	42	46.0	‑	6.93	4.31
160			23	21	16	20.0	‑	3.61	1.88
50			19	18	15	17.3	‑	2.08	1.63
16			9	13	13	11.7	‑	2.31	1.09
Positive reference control (2AA)(2 µg/plate)			278	260	232	256.7	‑	23.18	24.06

Obs : Observation (made by naked eye)                     TIP  : Test Item Particles

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.

 

 

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 typhimuriumTA98, TA100, TA1535,

                               TA1537;E. coli:Escherichia coliWP2uvrA

                                               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 was determined to be mutagenic in Salmonella typhimurium TA100 and TA1535 strains in the absence and presence of an exogenous metabolic activation system.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD guideline 471. The initial mutation test was carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) 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 TA100 and TA1535. The study included preliminary solubility tests, a preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (repeated plate incorporation test).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 a correction of the concentrations for the active component content (77.31 %) 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: 5000;1600; 500; 160; 50 and 16 µg/plate. The selection of the concentration range was based on the recommendations in the OECD guideline 471. 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 same concentration levels were investigated in the confirmatory mutation test: ±S9 mix: 5000; 1600; 500; 160; 50 and 16 µg/plate. When evaluated by naked eye, non-interfering test item precipitate (test item particles) was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 µg/plate in the absence and presence of an exogenous metabolic activation (±S9 mix) system. The test item did not show unequivocal inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) were considered to be within the biological variability range of the applied test system. The revertant colony numbers of solvent control plates (DMSO) 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, 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 test) in the investigated Salmonella typhimuriumTA100 and TA1535 strains in the absence and presence of the exogenous metabolic activation system (±S9 mix). The obtained revertant colony number increases were clearly above the corresponding historical control data ranges and the relevant genotoxicological threshold for being positive in both experiments. The increases were observed in strain TA100 at the highest examined concentration of 5000 µg/plate (±S9 mix) and in strain TA1535 in the concentration range of 5000-500 µg/plate (±S9 mix). The obtained increased tendencies, especially in the latter case, followed a clear dose-relationship. Therefore, the test item was determined to be mutagenic in Salmonella typhimurium TA100 and TA1535 strains carrying base pair substitutions, in the absence and presence of an exogenous metabolic activation system, under the test conditions used in this study.