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REACH

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

EC number: 288-914-8 | CAS number: 85940-25-6

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the bacterial reverse mutation assay (OECD 471), the test substance was considered to induced gene mutations in Salmonella typhimurium TA1537 with and without metabolic activation.

Based on the results of the mammalian cell mutagenicity test using Chinese hamster V79 cells (HPRT, OECD 476), the test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.

Based on the results of the in vitro micronucleus assay with Chinese hamster V79 cells (OECD 487), the test substance was considered to be non-mutagenic in Chinese hamster V79 cells.

In conclusion, based on a weight of evidence approach the test substance was considered not to be genotoxic.

Link to relevant study records

Referenceopen all close all

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

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. 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 / 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)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

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.

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

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

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

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

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.

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 10⁹CFU/mL

Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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 10⁹CFU/mL

Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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 10⁹CFU/mL

Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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

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 10⁹CFU/mL

Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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

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 10⁹CFU/mL

Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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

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 10⁹CFU/mL

Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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 10⁹CFU/mL

Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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 10⁹CFU/mL

Without Exogenous Metabolic Activation (-S9 mix)
Concentration (µg/plate)			Revertant per Plate			Mean	Obs	SD	MR
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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
Concentration (µg/plate)	Parallel:		1	2	3	Mean	Obs	SD	MR
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

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

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:

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.

Reference 2

Endpoint:: in vitro gene mutation study in mammalian cells
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: 19 December 2017 - 27 February 2018
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:: 2016
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:: 2008
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Type of assay:: mammalian cell gene mutation assay
Target gene:: HPRT
Species / strain / cell type:: Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):: - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:: with and without
Metabolic activation system:: Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:: with and without metabolic activation / 4 hours treatment: 1.0; 2.0; 3.9; 7.8; 15.6; 31.3; 62.5; and 125.0 µg/mL
The concentration range of the main experiment was based on precipitation observed in the pre-experiment and therefore 125 µg/mL was chosen as top concentration in the presence and absence of metabolic activation.
The concentrations of 1.0 to 15.6 µg/mL were evaluated for mutagenicity.
In the main experiment precipitation visible to the unaided eye was noted at concentrations of 15.6 µg/mL and above.
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: DMSO (1%)
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:: no
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: 7,12-dimethylbenzanthracene; ethylmethanesulphonate
Details on test system and experimental conditions:: METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp. 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:: A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a. at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. the increase is dose-related when evaluated with an appropriate trend test,
c. any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a. none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. there is no concentration-related increase when evaluated with an appropriate trend test,
c. all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.
Statistics:: A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was not performed since the mutation frequencies of all test points were within the 95% confidence interval.
However, both, biological and statistical significance were considered together.
: Key result
Species / strain:: Chinese hamster lung fibroblasts (V79)
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: no cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: not examined
Positive controls validity:: valid
Additional information on results:: TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.34 in the solvent control versus pH 7.44 at 500 µg/mL)
- Effects of osmolality: No relevant increase (463 mOsm in the solvent control versus 470 mOsm at 500 µg/mL)
- Evaporation from medium: Not examined
- Precipitation: determined at 15.6 µg/mL without metabolic activation and at 62.5 µg/mL with metabolic activation
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 3.9 µg/mL and 500 µg/mL were used with respect to the current OECD guideline 476, adopted 29 July 2016, regarding the solubility properties of the test item.
In the pre-experiment no relevant cytotoxicity was observed up to the highest concentration following 4 hours of treatment with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. At the beginning of treatment precipitation was observed at 125 µg/ml and above with and without metabolic activation. At the end of the 4 hours treatment precipitation occurred at 62.5 µg/mL and above with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The concentration range of the main experiment was based on precipitation observed in the pre-experiment and therefore 125 µg/mL was chosen as top concentration in the presence and absence of metabolic activation.
To overcome problems with possible deviations in toxicity and precipitation the main experiment was started with more than four concentrations. The cultures treated with the highest concentration of the test item with and without metabolic activation were not continued to avoid analysis of too many precipitating concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxic effects indicated by a relative adjusted cloning efficiency I below 50% occurred up to the maximum evaluated concentration with and without metabolic activation.
Conclusions:: In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
Executive summary:: The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD guideline 476. The cells were exposed to the test item for 4 hours with and without metabolic activation. The following concentrations were evaluated with and without metabolic activation: 1.0, 2.0, 3.9, 7.8 and 15.6 µg/mL. Precipitation of the test item was observed at the end of treatment at 15.6 µg/mL and above in the presence and absence of metabolic activation. No cytotoxic effects indicated by a relative adjusted cloning efficiency I below 50% occurred up to the maximum evaluated concentration with and without metabolic activation. No relevant increase in mutant colony numbers/1x10⁶cells was observed. The mean mutant frequency obtained in the solvent controls ranged from 12.8 to 14.7 mutants per 10⁶cells. The values were well within the 95% confidence interval of the laboratory historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. EMS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies. The range of the mean mutant frequencies of the groups treated with the test item ranged from 13.6 to 23.8 mutants per 10⁶cells. A linear regression analysis was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 January 2018 - 18 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

Qualifier:

according to guideline

Guideline:

other: EU Method B.49 (In Vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2017

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Concentrations:
- Experiment I (with and without S9): 2.3, 4.0, 7.0, 12.2, 21.3, 37.3, 65.3, 114, 200 and 500 µg/mL
- Experiment II: (without S9): 1.0, 2.0, 4.0, 8.0, 12.1, 14.5, 17.4, 20.8, 25.0, 50.0 µg/mL
- Experiment II: (with S9): 1.0, 2.0, 4.0, 8.0, 12.1, 14.5 µg/mL

Justification for top dose: chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline 487

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: chosen due to its solubility properties and its relative non-toxicity to the cell cultures

Untreated negative controls:

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Griseofulvin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: approximately 5.0 – 6.0 x 10^5 cells

DURATION
- Exposure duration: 4 hours (experiment I with and without S9 mix, experiment II with S9 mix) and 24 hours (experiment I without S9 mix)
- Expression time (cells in growth medium): 20 hours (experiment I with and without S9 mix, experiment II with S9 mix) and 0 hours (experiment I without S9 mix)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cells were detached by trypsin-EDTA-solution for approx. 5 minutes, followed by stopping the enzymatic treatment by adding complete culture medium including 10 % (v/v) FBS. The cultures were harvested and spun down by gentle centrifugation for 7 min. The supernatant was discarded and the cells were resuspended in saline G and spun down once again by centrifugation. Then the cells were resuspended in KCL solution (0.4 %) and incubated at 37°C for 10 minutes. Ice-cold fixative mixture of methanol and glacial acetic acid (19+1 parts, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the supernatant after centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping a small amount of the cell suspension in fresh fixative on clean, wet microscope slides and allowed to dry. The mounted cells were Giemsa-stained and, after drying, covered with coverslips.
NUMBER OF CELLS EVALUATED: at least 1000 binucleate cells per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: CBPI was determined in 500 cells per culture

Evaluation criteria:

The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

Statistics:

script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics, to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: In experiment I, precipitation of the test item in the culture medium was observed at 65.3 μg/mL and above in the absence of S9 mix and at 7.0 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in experiment II, in the presence of S9 mix, at 12.1 μg/mL and above at the end of treatment. In experiment II, in the absence of S9 mix, no precipitation was observed.
- Definition of acceptable cells for analysis: The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 24 hrs after start of the exposure.

CYTOKINESIS BLOCK: Cytochalasin B ws added to the cell culture to ensure that there are binucleate cells to be evaluated for micronuclei.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: At least 1000 binucleate cells per culture were scored for cytogenetic damage.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Table1: Summary of results

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
Exposure period 4 hrs without S9 mix
I	24 hrs	Solvent control¹	1.91		1.00
		Positive control²	1.86	6.2	7.10^S
		7.0	1.79	13.8	0.60
		12.2	1.71	22.9	1.00
		21.3	1.43	52.6	1.35
Exposure period 24 hrs without S9 mix
II	24 hrs	Solvent control¹	1.96		0.50
		Positive control³	2.12	n.c.	3.10^S
		2.0	1.90	6.3	0.35
		4.0	1.84	12.0	0.45
		8.0	1.40	58.2	0.70
Exposure period 4 hrs with S9 mix
I	24 hrs	Solvent control^1#	2.03		1.38
		Positive control⁴	1.85	17.5	9.85^S
		2.3^#	2.02	1.6	1.78
		4.0^#	1.99	3.8	2.08^S
		7.0^P/^#	1.97	5.9	2.23^S
II	24 hrs	Solvent control¹	1.97		1.05
		Positive control⁴	1.87	11.2	11.45^S
		4.0	1.95	2.4	1.10
		8.0	1.94	3.7	1.45
		12.1^P	1.83	15.0	1.75

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

^P Precipitation occurred at the end of treatment

^S The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

¹ DMSO 1.0 % (v/v)
²MMC 0.1 µg/mL

³Griseofulvin 6.0 µg/mL

⁴CPA 2.0 µg/mL

Table 2: Percentage of micronucleated cells in Chinese hamster V79 cultures (2009-2017), Aqueous solvents: DMEM/Ham’s F12, Deionised water (10 % v/v), Organic solvents: DMSO (0.5 or 1.0 %), Acetone, Ethanol and THF (0.5 %)

Solvent Control without S9
Micronucleated cells in %
	Pulse treatment (4/24)	Continuous treatment (24/24)	Continuous treatment (24/44)
No. of experiments	30	28	24
Mean	1.23	0.84	1.04
95 % Ctrl limit	0.00 - 2.56	0.00 - 1.72	0.00 - 2.21
1x SD (2x SD)	0.66 (1.33)	0.44 (0.88)	0.59 (1.18)
Min - Max	0.05–2.85	0.30–2.05	0.30–2.55

Solvent Control with S9
Micronucleated cells in %
	Pulse treatment (4/24)	Pulse treatment (4/44)
No. of experiments	36	19
Mean	0.99	0.79
95 % Ctrl limit	0.00 - 2.03	0.01 - 1.57
1x SD (2x SD)	0.52 (1.04)	0.39 (0.78)
Min - Max	0.20–2.35	0.15–1.75

Positive Control without S9
Micronucleated cells in %
	Pulse treatment (4/24)	Continuous treatment (24/24)	Continuous treatment (24/44)
	MMC	Griseofulvin	Griseofulvin
No. of experiments	22	15	13
Mean	11.46	8.28	9.42
95 % Ctrl limit	0.99 - 21.92	0.59 - 15.96	0.00 - 29.38
1x SD (2x SD)	5.23 (10.46)	3.84 (7.68)	9.98 (19.96)
Min - Max	5.70 – 28.10	2.90 – 15.50	4.10 – 39.80

Positive Control with S9
Micronucleated cells in %
	Pulse treatment (4/24)	Pulse treatment (4/44)
	CPA
No. of experiments	23	10
Mean	9.49	13.96
95 % Ctrl limit	0.00 – 19.13	0.00 - 32.64
1x SD (2x SD)	4.82 (9.64)	9.34 (18.68)
Min - Max	2.90 – 25.45	5.15 – 33.75

Conclusions:

The test item is considered to be non-mutagenic in this in vitro micronucleus test.

Executive summary:

The test item suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In experiment I, the exposure period was 4 hours with and without S9 mix. In experiment II, the exposure periods were 4 hours with S9 mix and 24 hours without S9 mix. The cells were prepared 24 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. At least 1000 cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined. The highest treatment concentration in this study, 500 μg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline 487 for thein vitromammalian cell micronucleus test.

In experiment I, precipitation of the test item in the culture medium was observed at 65.3 μg/mL and above in the absence of S9 mix and at 7.0 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in experiment II, in the presence of S9 mix, at 12.1 μg/mL and above at the end of treatment. In experiment II, in the absence of S9 mix, no precipitation was observed. No relevant influence on osmolarity or pH was observed. The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect however, is based on a final concentration of 1% DMSO in medium. As the osmolarity is measured by freezing point reduction, 1% of DMSO has a substantial impact on the determination of osmolarity. In the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentrations. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In experiment I in the presence of S9 mix, however, the values of the two highest evaluated concentrations (2.08 and 2.23% micronucleated cells) were statistically significantly increased and exceeded the range of the 95% control limit of the historical control data (0.00 – 2.03% micronucleated cells). Dose dependency tested by trend test was statistically significant (p-value: 0.039). In the confirmatory experiment II, in the presence of S9 mix, none of the values exceeded the range of the 95% control limit of the historical control data (0.00 – 2.03% micronucleated cells) nor were they statistically significantly increased. Although dose dependency tested by trend test was statistically significant (p-value: 0.034) the finding in experiment I can be regarded as biologically irrelevant. In both experiments, either Griseofulvin (6.0μg/mL), MMC (0.1 μg/mL) or CPA (2.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

Bacterial reverse mutation assay (OECD 471):

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.

Mammalian cell muation assay - HPRT (OECD 476):

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD guideline 476. The cells were exposed to the test item for 4 hours with and without metabolic activation. The following concentrations were evaluated with and without metabolic activation: 1.0, 2.0, 3.9, 7.8 and 15.6 µg/mL. Precipitation of the test item was observed at the end of treatment at 15.6 µg/mL and above in the presence and absence of metabolic activation. No cytotoxic effects indicated by a relative adjusted cloning efficiency I below 50% occurred up to the maximum evaluated concentration with and without metabolic activation. No relevant increase in mutant colony numbers/1x10⁶ cells was observed. The mean mutant frequency obtained in the solvent controls ranged from 12.8 to 14.7 mutants per 10⁶ cells. The values were well within the 95% confidence interval of the laboratory historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. EMS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies. The range of the mean mutant frequencies of the groups treated with the test item ranged from 13.6 to 23.8 mutants per 10⁶ cells. A linear regression analysis was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

In vitro micronucleus assay (OECD 487):

The test item suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In experiment I, the exposure period was 4 hours with and without S9 mix. In experiment II, the exposure periods were 4 hours with S9 mix and 24 hours without S9 mix. The cells were prepared 24 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. At least 1000 cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined. The highest treatment concentration in this study, 500μg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline 487 for thein vitromammalian cell micronucleus test.

In experiment I, precipitation of the test item in the culture medium was observed at 65.3 μg/mL and above in the absence of S9 mix and at 7.0 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in experiment II, in the presence of S9 mix, at 12.1 μg/mL and above at the end of treatment. In experiment II, in the absence of S9 mix, no precipitation was observed. No relevant influence on osmolarity or pH was observed. The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect however, is based on a final concentration of 1% DMSO in medium. As the osmolarity is measured by freezing point reduction, 1% of DMSO has a substantial impact on the determination of osmolarity. In the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentrations. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In experiment I in the presence of S9 mix, however, the values of the two highest evaluated concentrations (2.08 and 2.23% micronucleated cells) were statistically significantly increased and exceeded the range of the 95% control limit of the historical control data (0.00 – 2.03% micronucleated cells). Dose dependency tested by trend test was statistically significant (p-value: 0.039). In the confirmatory experiment II, in the presence of S9 mix, none of the values exceeded the range of the 95% control limit of the historical control data (0.00 – 2.03% micronucleated cells) nor were they statistically significantly increased. Although dose dependency tested by trend test was statistically significant (p-value: 0.034) the finding in experiment I can be regarded as biologically irrelevant. In both experiments, either Griseofulvin (6.0 μg/mL), MMC (0.1 μg/mL) or CPA (2.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Discussion and Conclusion:

The test item showed a mutagenic activity on Salmonella typhimurium TA1537 strains in the absence and presence of an exogenous metabolic activation system. The substance is a high molecular weight UVCB compound with number-average molecular weight of 96 601 Da and weight-average molecular weight of 189 317 714 Da and no fraction with a molecular weight below 1000 Da. Thus, it is highly unlikely that the substance can pass intact cell membranes. In contrast to mammalian cells the S. typhimurium strains used in the bacterial reverse mutation assay have a rfa mutation which enhances the permeability of the cell membrane. It was assumed that the mutagenic result obtained with the test substance in tester strain TA1537 is a result of the impaired barrier function of the bacterial cell wall and not relevant for intact organisms. In order to confirm the assumption an HPRT and micronucleus test in Chinese hamster V79 cells according to OECD guideline 476 and 487, respectively, were performed. These two tests showed no genotoxic activity of the test substance. Therefore, based on a weight of evidence approach the test substance was considered not to be genotoxic.

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 twelfth time in Regulation (EC) No 2019/521.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

				relative	relative	rel. adjusted	mutant	95%
	conc.	P	S9	cloning	cell	cloning	colonies/	confidence
	µg/mL		mix	efficiency I	density	efficiency I	10⁶cells	interval
				%	%	%
Solvent control with DMSO			-	100.0	100.0	100.0	12.8	1.7 - 30.2
Positive control (EMS)	300.00		-	79.8	96.8	77.2	406.8	1.7 - 30.2
Test item	1.0		-	74.1	100.0	73.9	16.6	1.7 - 30.2
Test item	2.0		-	115.1	82.9	94.6	13.6	1.7 - 30.2
Test item	3.9		-	87.4	101.2	87.2	20.3	1.7 - 30.2
Test item	7.8		-	107.7	83.1	89.2	16.8	1.7 - 30.2
Test item	15.6	P	-	104.0	77.4	79.1	20.2	1.7 - 30.2
Test item	31.3	P	-	#	#	#	#	#
Test item	62.5	P	-	#	#	#	#	#
Test item	125.0	P	-	#	#	#	#	#
Solvent control with DMSO			+	100.0	100.0	100.0	14.7	2.0 - 29.4
Positive control (DMBA)	2.3		+	95.8	86.7	82.9	143.5	2.0 - 29.4
Test item	1.0		+	95.6	96.5	92.4	21.3	2.0 - 29.4
Test item	2.0		+	97.5	82.5	80.5	21.9	2.0 - 29.4
Test item	3.9		+	99.5	98.6	98.1	22.4	2.0 - 29.4
Test item	7.8		+	100.2	78.0	78.3	23.8	2.0 - 29.4
Test item	15.6	P	+	97.3	79.6	77.4	19.4	2.0 - 29.4
Test item	31.3	P	+	#	#	#	#	#
Test item	62.5	P	+	#	#	#	#	#
Test item	125.0	P	+	#	#	#	#	#

Registration Dossier

Registration Dossier

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Diss Factsheets

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

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

Referenceopen all close all

Reference 1

Reference 2

Summary of Results of the main experiment

Mean Values of Culture I and II

Reference 3

Endpoint conclusion

Genetic toxicity in vivo

Endpoint conclusion

Additional information

Justification for classification or non-classification

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

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

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Summary of Results of the main experiment

Mean Values of Culture I and II

Reference 3

Endpoint conclusion

Genetic toxicity in vivo

Endpoint conclusion

Additional information

Justification for classification or non-classification

Referenceopen all close all