Phenol, 4-amino-, reaction products with aniline, 4-methyl-1,3-benzenediamine, p-phenylenediamine, sodium sulfide (Na2S) and sulfur

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of an OECD 471 study, the test substance is considered to be non-mutagenic with and without metabolic activation in bacteria.

Based on the results of an OECD 473 study, the read across substance (CAS: 90268 -98 -7) is considered to be non-clastogenic with and without metabolic activation in Chinese Hamster lung V79 cells.

Based on the results of an OECD 476 study, the read across substance (CAS: 90268 -98 -7) is considered to be non-mutagenic with and without metabolic activation in Chinese hamster ovary cells.

Link to relevant study records

Reference

Reference 1

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 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

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

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. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver

Test concentrations with justification for top dose:

5000, 1600, 500, 160, 50, 16 and 5 µg/plate

Vehicle / solvent:

dimethyl sulfoxide

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..

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 11-14 hours in a 37 °C 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 was done on the basis of solubility tests and concentration range finding tests (informatory toxicity tests)

Evaluation criteria:

The colony numbers on the controls (untreated, vehicle, positive) and the test plates were determined (counted manually), the mean values and appropriate standard deviations and 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.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: 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				WP2 uvrA
	-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	12.7	0.73	16.7	0.57	50.7	0.69	77.3	0.95	9.0	0.71	11.7	1.25	8.3	0.78	5.7	0.52	21.7	0.70	26.3	0.53
1600	18.3	1.06	21.7	0.75	74.7	1.02	83.0	1.02	9.0	0.71	6.7	0.71	6.7	0.63	7.7	0.70	32.3	1.04	30.0	0.61
500	17.3	1.00	29.7	1.02	81.3	1.11	91.0	1.12	11.0	0.87	10.7	1.14	9.0	0.84	10.3	0.94	34.3	1.11	45.3	0.92
160	25.3	1.46	24.7	0.85	97.7	1.34	88.3	1.09	12.3	0.97	8.0	0.86	12.0	1.13	10.0	0.91	32.0	1.03	38.7	0.78
50	24.0	1.38	22.0	0.76	94.0	1.29	99.0	1.22	13.3	1.05	8.7	0.93	10.0	0.94	9.7	0.88	28.7	0.92	46.0	0.93
16	32.7	1.88	27.7	0.95	92.0	1.26	95.0	1.17	12.0	0.95	8.0	0.86	14.3	1.34	13.7	1.24	35.7	1.15	34.7	0.70
5	32.7	1.88	24.3	0.84	78.0	1.07	110.7	1.37	11.3	0.89	9.7	1.04	13.3	1.25	12.3	1.12	25.7	0.83	39.0	0.79
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.

Table 2: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2 uvrA
	-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	22.0	1.29	30.7	1.18	81.0	1.03	100.3	1.07	13.7	0.75	10.7	1.10	11.0	1.14	10.0	1.20	32.7	1.10	43.0	0.90
DMSO Control	17.0	1.00	26.0	1.00	79.0	1.00	93.7	1.00	18.3	1.00	9.7	1.00	9.7	1.00	8.3	1.00	29.7	1.00	48.0	1.00
Ultrapure Water Control	–	–	–	–	92.0	1.00	–	–	17.7	1.00	–	–	–	–	–	–	42.0	1.00	–	–
5000	5.3	0.31	7.0	0.27	57.0	0.72	71.3	0.76	8.3	0.45	6.3	0.66	6.0	0.62	7.3	0.88	23.7	0.80	29.0	0.60
1600	13.0	0.76	16.0	0.62	69.3	0.88	78.0	0.83	10.7	0.58	8.7	0.90	7.0	0.72	9.0	1.08	18.3	0.62	36.0	0.75
500	22.0	1.29	20.3	0.78	76.7	0.97	93.0	0.99	13.3	0.73	9.7	1.00	9.0	0.93	8.0	0.96	19.0	0.64	42.3	0.88
160	17.7	1.04	25.3	0.97	74.3	0.94	86.0	0.92	15.0	0.82	9.3	0.97	10.7	1.10	8.7	1.04	26.7	0.90	34.7	0.72
50	16.7	0.98	31.0	1.19	72.3	0.92	96.3	1.03	14.3	0.78	11.3	1.17	11.0	1.14	7.7	0.92	27.3	0.92	43.0	0.90
16	24.0	1.41	27.7	1.06	66.7	0.84	98.7	1.05	12.0	0.65	12.0	1.24	11.0	1.14	9.0	1.08	35.3	1.19	44.7	0.93
5	18.7	1.10	25.7	0.99	91.0	1.15	94.0	1.00	15.7	0.85	12.3	1.28	8.3	0.86	12.3	1.48	30.0	1.01	50.0	1.04
NPD (4mg)	252.0	14.82	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1050.7	11.42	–	–	1605.3	90.87	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	564.0	58.34	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	2101.3	50.03	–	–
2AA (2mg)	–	–	2008.0	77.23	–	–	2178.7	23.26	–	–	161.0	16.66	–	–	129.0	15.48	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	196.0	4.08

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.

Table 3: 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

Table 4: 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

Conclusions:

The test item has no mutagenic activity on the applied bacterium tester strains 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 according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The study included a preliminary solubility test, two preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item suspensions/solutions a correction of the concentrations for the active component (dyestuff) content (9.4 %) was made in the experiments.

Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50; 16 and 5 μg dyestuff/plate (corresponding to 53200, 17024, 5320, 1702, 532, 170, 53 µg product/plate). The selection of the concentration range was based on the recommendations in OECD 471 guideline. At the concentration choice the slight toxicity of the test item and the precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye.

When evaluated by naked eye, non-interfering test item precipitate or appearance of test item particles was noticed after about 48 hours incubation on the plates in the examined strains in the concentration range of 5000-500 μg dyestuff/plate in the absence and presence of an exogenous metabolic activation system following the plate incorporation procedure and in the absence of an exogenous metabolic activation system following the pre-incubation procedure. Furthermore non-interfering test item precipitate or appearance of test item particles was noticed at 5000 and 1600 μg dyestuff/plate in the presence of an exogenous metabolic activation system following the pre-incubation procedure. A slight, but unequivocal inhibitory effect of the test item was observed in the initial mutation test in the S. typhimurium strain TA100 at the highest examined concentration of 5000 μg dyestuff/plate in the absence of an exogenous metabolic activation system. In the confirmatory mutation test this effect was observed in the S. typhimurium strains TA98 and TA100 at 5000 μg dyestuff/plate in the absence and in S. typhimurium TA98 also in the presence of an exogenous metabolic activation system. The inhibitory effect was indicated by decreased revertant colony counts (below the corresponding historical control data range). The background lawn development was not affected in any case. All of the further 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 dimethyl sulfoxide (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges (or even were above the range), thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment withthe test itemat any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Please refer to the attached read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Please refer to the attached read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

Sub-line (K1)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

evidence of toxicity was seen at the highest tested concentration with the test item in presence and absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The study included a preliminary solubility test, two preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item suspensions/solutions a correction of the concentrations for the active component (dyestuff) content (9.4 %) was made in the experiments. Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50; 16 and 5 μg dyestuff/plate (corresponding to 53200, 17024, 5320, 1702, 532, 170, 53 µg product/plate). The selection of the concentration range was based on the recommendations in OECD 471 guideline. At the concentration choice the slight toxicity of the test item and the precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye. When evaluated by naked eye, non-interfering test item precipitate or appearance of test item particles was noticed after about 48 hours incubation on the plates in the examined strains in the concentration range of 5000-500 μg dyestuff/plate in the absence and presence of an exogenous metabolic activation system following the plate incorporation procedure and in the absence of an exogenous metabolic activation system following the pre-incubation procedure. Furthermore non-interfering test item precipitate or appearance of test item particles was noticed at 5000 and 1600 μg dyestuff/plate in the presence of an exogenous metabolic activation system following the pre-incubation procedure. A slight, but unequivocal inhibitory effect of the test item was observed in the initial mutation test in the S. typhimurium strain TA100 at the highest examined concentration of 5000 μg dyestuff/plate in the absence of an exogenous metabolic activation system. In the confirmatory mutation test this effect was observed in the S. typhimurium strains TA98 and TA100 at 5000 μg dyestuff/plate in the absence and in S. typhimurium TA98 also in the presence of an exogenous metabolic activation system. The inhibitory effect was indicated by decreased revertant colony counts (below the corresponding historical control data range). The background lawn development was not affected in any case. All of the further 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 dimethyl sulfoxide (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges (or even were above the range), thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment withthe test itemat any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

OECD 473:

The read across substance (CAS: 90268 -98 -7) suspended in DMSO was tested in a chromosome aberration assay in V79 cells in two independent experiments according to OECD guideline 473. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix): 15.6, 31.3, 62.5, 125 and 180 µg/mL test item for Experiment A with 3/20 h treatment/sampling time and Experiment B with 3/28 h treatment/sampling time and 3.9, 7.8, 15.6, 31.3 and 45 µg/mL test item for Experiment B with 20/20 h and 20/28 h treatment/sampling time. Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the absence and presence of metabolic activation and in experiment B in the presence of metabolic activation, some values were slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationships were observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 L/mL) and cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the test item is considered as being non-clastogenic in this system.

OECD 476:

The read across substance (CAS: 90268 -98 -7) suspended in DMSO was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD guideline 476. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver: 62.5, 125, 250, 350 and 450 µg/mL (without S9-mix) and 125, 250, 500, 750 and 1000 µg/mL (with S9-mix). In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity. After the 5-hour treatment period phenotypic expression was evaluated up to 8 days following exposure. In the absence and presence of metabolic activation clear cytotoxicity (survival approximately 16%) of the test item was observed at the highest concentration applied (450 µg/mL in the absence and 1000 µg/mL in the presence of S9 mix). In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. In the absence of S9 mix, in the cultures treated with 350 and 450 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 4 of 4 cultures, respectively). In the presence of S9 mix, in the cultures treated with 500 and 750 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 2 of 4 cultures, respectively). These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls and the historical control range were observed. The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulfonate (1.0 µL/mL) and 7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.