Phenol, 2,4-dinitro-, sulfurized, thiosulfonated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In an in vitro chromosome aberration assay (CA) according to OECD guideline 473, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

In an in vitro mammalian gene mutation test (HPRT) in CHO-K1 cells according to OECD guideline 476, the test item did not show mutagenic properties.

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:

21 June - 09 August, 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:

21 July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August, 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Date of production: 30.11.2016
Expiration date: 30.11.2021

Target gene:

his/trp
In addition to histidine (his) or tryptophan (trp) 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:

S9 mix of phenobarbital and β-naphthoflavone-induced rat liver

Test concentrations with justification for top dose:

5000, 1600, 500, 160, 50, and 16 µg/plate
Selection of the concentration range was done on the basis of a solubility test and a concentration range finding test (informatory toxicity test).

Vehicle / solvent:

- Solvents used: ultrapure water (ASTM Type I), Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent: In the study two types of solvent control were used depending on the solubility of the test item and the solubility of positive control reference items.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine

Remarks:

TA98, without S9 mix, 4 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537, without S9 mix, 50 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

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

Details on test system and experimental conditions:

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

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

NUMBER OF REPLICATIONS:
3

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies, background lawn growth

Rationale for test conditions:

Based on the solubility test, a stock solution/suspension with a concentration of 50 mg/mL was prepared in ultrapure water (ASTM Type I) and diluted accordingly. In the informatory toxicity test a correction factor, based on the active component of the test item (80.2 %) was taken into consideration.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
In the informatory toxicity test the revertant colony numbers of solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected biological relevant increases in induced revertant colonies in both tester strains.

Evaluation criteria:

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



* : untreated, solvent or positive control

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the 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 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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

Table 4: 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	21.0	1.03	26.0	1.18	93.7	1.05	96.7	1.13	8.3	0.89	11.3	0.87	7.0	0.81	8.3	1.09	26.7	1.00	25.7	0.99
DMSO Control	22.7	1.00	24.3	1.00	–	–	88.3	1.00	–	–	8.7	1.00	8.3	1.00	8.0	1.00	–	–	34.7	1.00
Ultrapure Water Control	20.3	1.00	22.0	1.00	89.0	1.00	85.7	1.00	9.3	1.00	13.0	1.00	8.7	1.00	7.7	1.00	26.7	1.00	26.0	1.00
5000	16.7	0.82	12.0	0.55	69.3	0.78	84.7	0.99	17.0	1.82	18.0	1.38	9.0	1.04	7.3	0.96	30.3	1.14	20.7	0.79
1600	15.3	0.75	14.0	0.64	77.7	0.87	82.0	0.96	8.0	0.86	9.7	0.74	11.0	1.27	10.3	1.35	25.0	0.94	20.3	0.78
500	17.0	0.84	15.3	0.70	80.0	0.90	89.7	1.05	8.7	0.93	10.3	0.79	7.7	0.88	9.3	1.22	29.3	1.10	21.0	0.81
160	16.3	0.80	23.3	1.06	72.3	0.81	90.0	1.05	7.3	0.79	11.0	0.85	9.3	1.08	6.3	0.83	30.7	1.15	26.3	1.01
50	17.3	0.85	29.0	1.32	72.3	0.81	83.3	0.97	10.7	1.14	14.0	1.08	11.7	1.35	6.7	0.87	28.0	1.05	28.3	1.09
16	18.0	0.89	25.7	1.17	80.3	0.90	94.0	1.10	8.3	0.89	11.7	0.90	6.7	0.77	6.0	0.78	27.0	1.01	32.0	1.23
NPD (4mg)	306.0	13.50	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	988.0	11.10	–	–	1428.0	153.00	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	209.0	25.08	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	810.7	30.40	–	–
2AA (2mg)	–	–	1957.3	80.44	–	–	1001.3	11.34	–	–	222.3	25.65	–	–	191.3	23.92	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	202.0	5.83

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

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

Table 5 : 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	18.3	1.31	20.7	1.09	82.7	1.06	102.3	1.07	15.0	1.05	12.3	1.00	7.0	1.40	9.0	0.93	29.3	0.75	32.7	0.82
DMSO Control	14.7	1.00	20.0	1.00	–	–	92.0	1.00	–	–	14.7	1.00	6.0	1.00	8.7	1.00	–	–	37.3	1.00
Ultrapure Water Control	14.0	1.00	19.0	1.00	77.7	1.00	95.7	1.00	14.3	1.00	12.3	1.00	5.0	1.00	9.7	1.00	39.3	1.00	40.0	1.00
5000	13.0	0.93	14.7	0.77	55.0	0.71	88.3	0.92	17.0	1.19	24.3	1.97	4.7	0.93	7.0	0.72	33.3	0.85	32.0	0.80
1600	12.3	0.88	17.0	0.89	77.7	1.00	80.7	0.84	15.7	1.09	13.3	1.08	6.0	1.20	8.7	0.90	28.0	0.71	36.0	0.90
500	13.7	0.98	21.7	1.14	72.0	0.93	78.3	0.82	12.0	0.84	11.3	0.92	5.0	1.00	10.3	1.07	34.0	0.86	30.3	0.76
160	15.3	1.10	21.3	1.12	77.3	1.00	79.3	0.83	10.7	0.74	12.7	1.03	6.7	1.33	10.7	1.10	46.3	1.18	40.3	1.01
50	15.3	1.10	23.7	1.25	68.3	0.88	89.7	0.94	10.3	0.72	14.7	1.19	5.0	1.00	10.7	1.10	38.0	0.97	33.7	0.84
16	17.7	1.26	31.3	1.65	74.7	0.96	100.7	1.05	11.7	0.81	13.3	1.08	5.0	1.00	7.3	0.76	32.0	0.81	48.0	1.20
NPD (4mg)	304.0	20.73	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	981.3	12.64	–	–	978.7	68.28	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	738.7	123.11	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1544.0	39.25	–	–
2AA (2mg)	–	–	1411.3	70.57	–	–	2442.7	26.55	–	–	234.0	15.95	–	–	209.7	24.19	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	249.7	6.69

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

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

Table 6: 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

			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:

In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (Ames) 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 S9 mix prepared from livers of phenobarbital/beta-naphthoflavone-induced rats. The study included preliminary solubility test, preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was in ultrapure water (ASTM Type I). At the formulation of test item solutions correction of concentrations for active component content (80.2 % dyestuff) was made in the main experiments. Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item (based on 97.4 % dyestuff) were prepared and investigated in the initial and confirmatory mutation tests: 5000, 1600, 500, 160, 50, and 16 µg/plate. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study. The test item did not show unequivocal inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) were considered to be within the biological variability range of the applied test system. The revertant colony numbers of solvent control (ultrapure water (ASTM Type I)) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was solved in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected biologically relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test 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:

experimental study

Adequacy of study:

key study

Study period:

15 September, 2017 - 03 January, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29 July, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

14 February, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: structural chromosome aberrations test in somatic and/or germ cells

Specific details on test material used for the study:

Expiration date: 30 Nov 2021

Target gene:

structural chromosome aberrations in metaphase somatic and/or germ cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cells Cultures), Lot. No.: 10H016
- Suitability of cells: The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates
- Doubling time 12 - 14 h
- Sex: male
- Methods for maintenance in cell culture: The cell stocks were kept in liquid nitrogen and were routinely checked for mycoplasma infections. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm² plastic flasks at 37 ± 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2.
- Modal number of chromosomes: diploid number, 2n=22

MEDIA USED
- Type and identity of media including CO2 concentration: DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1% of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%). 37 °C, 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: not specified

Cytokinesis block (if used):

colchicine (0.2 μg/mL)

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:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 39.1, 78.2, 156.3 and 312.5 µg/mL test item
with S9 mix: 39.1, 78.2, 156.3 and 312.5 µg/mL test item

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 39.1, 78.2, 156.3 and 312.5 µg/mL test item

Vehicle / solvent:

- Solvent used: DME (Dulbecco’s Modified Eagle’s)
- Justification for choice of solvent: DME is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Experiment A, without S9 mix, 1.0 µL/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Experiment B, without S9 mix, 0.4 µL/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Experiment A + B, with S9 mix, 5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10E5

DURATION
- Preincubation period:
- Exposure duration: 3 h
- Expression time: 20 h
- Fixation time: 10 min. in 3:1 mixture of methanol: acetic-acid

SPINDLE INHIBITOR (cytogenetic assays):
Cell cultures were treated with colchicine (0.2 μg/mL) 2.5 hours prior to harvesting.

STAIN (for cytogenetic assays):
The preparation was stained with 5 % Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300; 150 per duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)

Evaluation criteria:

Evaluation of Result
- The percentage of cells with structural chromosome aberration(s) was evaluated.
- Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
- Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
- Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
- Individual culture data were summarised in tabular form.
- There were no equivocal results in this study.
-pH and Osmolality data were summarised in tabular form.

Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is dose-related when evaluated with an appropriate trend test
- any of the results are outside the distribution of the laboratory historical negative control data

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the laboratory historical negative control data

Both biological and statistical significance should be considered together.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

For statistical analysis CHI² test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and
number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the
concurrent negative control.
The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95%
confidence intervals of historical control were calculated with C-chart.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH or osmolality: There were no relevant changes in pH or osmolality after treatment with the test item.
- Water solubility: A clear solution of Solubilised Sulphur Black 1 was obtained in DME (Dulbecco’s Modified Eagle’s) medium up to a concentration of 25 mg/mL.
- Precipitation: There was no precipitation in the medium at any concentration tested.

RANGE-FINDING/SCREENING STUDIES:
A pre-test on cytotoxicity was performed as part of this study to establish an appropriate concentration range for the main chromosome aberration assays (experiment A and B), both in the absence and in the presence of a metabolic activation (rodent S9 mix). Based on cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. Based on the results of the cytotoxicity assay the following concentrations were selected for the chromosome aberration assay:

HISTORICAL CONTROL DATA
- Positive historical control data:
Without S9 mix:
Ethyl methanesulfonate (excl. gaps): Mean 31.47; SD 3.77; Lower confidence interval 23.51; upper confidence interval 39.44
With S9 mix:
Cyclophasphamide (excl. gaps): Mean 39.39; SD 2.62; Lower confidence interval 33.85; upper confidence interval 44.93

- Negative (solvent/vehicle) historical control data:
Without S9 mix (excl. gaps): Mean 2.75; SD 0.65; Lower confidence interval 1.39; upper confidence interval 4.11
With S9 mix (excl. gaps): Mean 2.86; SD 0.65; Lower confidence interval 1.50; upper confidence interval 4.22

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

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

Table 4: 3-hour treatment without and with S9 mix / 20-hour sampling time 

Test group	Concentration (µg/mL)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	2050000	2000000	1975000	-	-	-
	-	B	–	1900000	1900000
	-	C	–	2100000	1850000
	-	D	–	1950000	2050000
Solvent control (DME)	-	A	–	7400000	7250000	7325000	5350000	100.00	0.00
	-	B	–	7350000	7300000
Solubilised Sulphur Black 1	39.1	A	–	7200000	7050000	7125000	5150000	96.26	3.74
	78.2	A	–	6250000	6550000	6400000	4425000	82.71	17.29
	156.3	A	–	5650000	5900000	5775000	3800000	71.03	28.97
	312.5	A	–	4500000	4250000	4375000	2400000	44.86	55.14
	625	A	–	3750000	3500000	3625000	1650000	30.84	69.16
	1250	A	–	3100000	2950000	3025000	1050000	19.63	80.37
	2500	A	–	850000	1000000	925000	-1050000*	-19.63**	119.63***
	5000	A	–	0	0	0	-1975000*	-36.92**	136.92***
	EMS 1 µL/mL	A	–	4850000	4600000	4725000	2750000	51.40	48.60
Solvent control (DME)	-	A	+	7500000	7650000	7575000	5600000	100.00	0.00
	-	B	+	7400000	7750000
Solubilised Sulphur Black 1	39.1	A	+	7500000	7500000	7500000	5525000	98.66	1.34
	78.2	A	+	6050000	5750000	5900000	3925000	70.09	29.91
	156.3	A	+	5350000	5450000	5400000	3425000	61.16	38.84
	312.5	A	+	4750000	4550000	4650000	2675000	47.77	52.23
	625	A	+	4150000	3850000	4000000	2025000	36.16	63.84
	1250	A	+	2600000	2450000	2525000	550000	9.82	90.18
	2500	A	+	1000000	1200000	1100000	-875000*	-15.63**	115.63***
	5000	A	+	0	0	0	-1975000*	-35.27**	135.27***
	Cycl. 5µg/mL	A	+	4700000	4850000	4775000	2800000	50.00	50.00

RICC=Relative Increase in Cell Counts               EMS: Ethyl methanesulfonate (EMS)                    *: cell number decrease          

Cytotoxicity= 100-RICC                                        Cycl: Cyclophosphamide monohydra                    **: zero RICC value,             ***:100% cytotoxicity

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY 

Table 5: 20-hour treatment without S9 mix / 20-hour sampling time 

Test group	Concentration (µg/mL)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	2050000	2000000	1975000	-	-	-
	-	B	–	1900000	1900000
	-	C	–	2100000	1850000
	-	D	–	1950000	2050000
Solvent control (DME)	-	A	–	6100000	6250000	6237500	4262500	100.00	0.00
	-	B	–	6200000	6400000
Solubilised Sulphur Black 1	39.1	A	–	4650000	4500000	4575000	2600000	61.00	39.00
	78.2	A	–	3000000	3000000	3000000	1025000	24.05	75.95
	156.3	A	–	1650000	1400000	1525000	-450000*	-10.56**	110.56***
	312.5	A	–	700000	500000	600000	-1375000*	-32.26**	132.26***
	625	A	–	50000	0	25000	-1950000*	-45.75**	145.75***
	1250	A	–	0	0	0	-1975000*	-46.33**	146.33***
	2500	A	–	0	0	0	-1975000*	-46.33**	146.33***
	5000	A	–	0	0	0	-1975000*	-46.33**	146.33***
	EMS 1 µL/mL	A	–	4100000	4300000	4200000	2225000	52.20**	47.80***

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY 

Table 6: 20-hour treatment without S9 mix and 3-hour treatment with S9 mix / 28-hour sampling time 

Test group	Concentration (µg/mL)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	2050000	2000000	1975000	-	-	-
	-	B	–	1900000	1900000
	-	C	–	2100000	1850000
	-	D	–	1950000	2050000
Solvent control (DME)	-	A	–	8950000	9050000	8987500	7012500	100.00	0.00
	-	B	–	9100000	8850000
Solubilised Sulphur Black 1	39.1	A	–	6150000	6150000	6150000	4175000	59.54	40.46
	78.2	A	–	3500000	3750000	3625000	1650000	23.53	76.47
	156.3	A	–	1500000	1800000	1650000	-325000*	-4.63**	104.63***
	312.5	A	–	450000	300000	375000	-1600000*	-22.82**	122.82***
	625	A	–	150000	50000	100000	-1875000*	-26.74**	126.74***
	1250	A	–	0	0	0	-1975000*	-28.16**	128.16***
	2500	A	–	0	0	0	-1975000*	-28.16**	128.16***
	5000	A	–	0	0	0	-1975000*	-28.16**	128.16***
	EMS 1 µL/mL	A	–	5900000	5600000	5750000	3775000	53.83**	46.17***
Solvent control (DME)	-	A	+	7750000	7850000	7812500	5837500	100.00	0.00
	-	B	+	7800000	7850000
Solubilised Sulphur Black 1	39.1	A	+	7500000	7500000	7500000	5525000	94.65	5.35
	78.2	A	+	6350000	6150000	6250000	4275000	73.23	26.77
	156.3	A	+	5500000	5700000	5600000	3625000	62.10	37.90
	312.5	A	+	4750000	4550000	4650000	2675000	45.82	54.18
	625	A	+	2400000	2200000	2300000	325000	5.57	94.43
	1250	A	+	1100000	1400000	1250000	-725000	-12.42	112.42
	2500	A	+	150000	0	75000	-1900000	-32.55	132.55
	5000	A	+	0	0	0	-1975000	-33.83	133.83
	Cycl. 5µg/mL	A	+	4950000	5000000	4975000	3000000	51.39	48.61

Table 7: Mean Number of Cells with structural Chromosome Aberrations, Experiment A

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
				incl. gaps	excl. gaps
Negative (Solvent) control	-	3 h	20 h	8	3
Solubilised Sulphur Black 1
39.1 µg/mL	-	3 h	20 h	9	3
78.2 µg/mL	-	3 h	20 h	9	3
156.3 µg/mL	-	3 h	20 h	9	3
312.5 µg/mL	-	3 h	20 h	10	3
Pos. Control (Ethyl methanesulphonate)	-	3 h	20 h	38**	30**
Negative (Solvent) control	+	3 h	20 h	8	4
Solubilised Sulphur Black 1
39.1 µg/mL	+	3 h	20 h	8	5
78.2 µg/mL	+	3 h	20 h	8	4
156.3 µg/mL	+	3 h	20 h	9	5
312.5 µg/mL	+	3 h	20 h	9	5
Pos. Control (Cyclophosphamide)	+	3 h	20 h	43**	37**

Positive control (-S9): Ethyl methanesulphonate (1.0 µL/mL)

Positive control (+S9): Cyclophosphamide (5.0 µg/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

Table 8: Mean Number of Cells with structural Chromosome Aberrations, Experiment B

Concentration (µg/mL)		S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
					incl. gaps	excl. gaps
	Negative (Solvent) control	-	20 h	20 h	8	3
	Solubilised Sulphur Black 1
	9.8 µg/mL	-	20 h	20 h	8	4
	19.6 µg/mL	-	20 h	20 h	8	3
	39.1 µg/mL	-	20 h	20 h	7	4
	58.7 µg/mL	-	20 h	20 h	8	4
	Pos. Control (Ethyl methanesulphonate)	-	20 h	20 h	45**	37**
	Negative (Solvent) control	-	20 h	28 h	8	3
	Solubilised Sulphur Black 1
	9.8 µg/mL	-	20 h	28 h	7	3
	19.6 µg/mL	-	20 h	20 h	9	4
	39.1 µg/mL	-	20 h	28 h	7	2
	58.7 µg/mL	-	20 h	28 h	9	4
	Pos. Control (Ethyl methanesulphonate)	-	20 h	28 h	45**	38**

Positive control (-S9): Ethyl methanesulphonate (0.4 µL/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

Table 9: Mean Number of Cells with structural Chromosome Aberrations, Experiment B 

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
				incl. gaps	excl. gaps
Negative (Solvent) control	+	3 h	28 h	8	4
Solubilised Sulphur Black 1
39.1 µg/mL	+	3 h	28 h	8	4
78.2 µg/mL	+	3 h	28 h	8	3
156.3 µg/mL	+	3 h	28 h	9	4
312.5 µg/mL	+	3 h	28 h	9	4
Pos. Control (Cyclophosphamide)	+	3 h	28 h	46**	40**

Cyclophosphamide: 5.0 µg/mL

** = p < 0.01 to the concurrent negative control and to the historical control

 

Table 10: Number of polyploidy Cells and endoreduplicated Cells, Experiment A 

Concentration (µg/mL)	S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	-	3/20 h	0.0	0.0
Solubilised Sulphur Black 1
39.1 µg/mL	-	3/20 h	0.0	0.0
78.2 µg/mL	-	3/20 h	0.0	0.0
156.3 µg/mL	-	3/20 h	0.0	0.0
312.5 µg/mL	-	3/20 h	0.0	0.0
Pos. Control (Ethyl methanesulphonate)	-	3/20 h	0.0	0.0
Negative (Solvent) control	+	3/20 h	0.0	0.0
Solubilised Sulphur Black 1
39.1 µg/mL	+	3/20 h	0.0	0.0
78.2 µg/mL	+	3/20 h	0.0	0.0
156.3 µg/mL	+	3/20 h	0.0	0.0
312.5 µg/mL	+	3/20 h	0.0	0.0
Pos. Control (Cyclophosphamide)	+	3/20 h	0.0	0.0

Ethyl methanesulphonate: 1.0 µmL/mL

Cyclophosphamide: 5.0 µg/mL

 The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

Table 11: Number of polyploidy Cells and endoreduplicated Cells, Experiment B 

Concentration (µg/mL)	S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	-	20/20 h	0.0	0.0
Solubilised Sulphur Black 1
9.8 µg/mL	-	20/20 h	0.0	0.0
19.6 µg/mL	-	20/20 h	0.0	0.0
39.1 µg/mL	-	20/20 h	0.0	0.0
58.7 µg/mL	-	20/20 h	0.0	0.0
Pos. Control	-	20/20 h	0.0	0.0
Negative (Solvent) control	-	20/28 h	0.0	0.0
Solubilised Sulphur Black 1
9.8 µg/mL	-	20/28 h	0.0	0.0
19.6 µg/mL	-	20/28 h	0.0	0.0
39.1 µg/mL	-	20/28 h	0.0	0.0
58.7 µg/mL	-	20/28 h	0.0	0.0
Pos. Control	-	20/28 h	0.0	0.0

Positive control (-S9):Ethyl methanesulphonate (0.4 µL/mL), The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

Table 12: Number of polyploidy Cells and endoreduplicated Cells, Experiment B

 

Concentration (µg/mL)	S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	+	3/28 h	0.0	0.0
Solubilised Sulphur Black 1
39.1 µg/mL	+	3/28 h	0.0	0.0
78.2 µg/mL	+	3/28 h	0.0	0.0
156.3 µg/mL	+	3/28 h	0.0	0.0
312.5 µg/mL	+	3/28 h	0.0	0.0
Pos. Control	+	3/28 h	0.0	0.0

Cyclophosphamide: 5.0 µg/mL

 

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

 

Table 13: pH AND Osmolality Data, Experiment A

 

Concentration (µg/mL)		pH	Osmolality (mmol/kg)
3-hour treatment without S9 Mix / 20-hour sampling time
Negative (solvent) Control		8.09	339
Solubilised Sulphur Black 1	39.1	8.34	350
	78.2	8.40	354
	156.3	8.32	347
	312.5	8.37	355
	EMS	8.32	349
3-hour treatment with S9 Mix / 20-hour sampling time
Negative (solvent) Control		8.17	345
Solubilised Sulphur Black 1	39.1	8.22	350
	78.2	8.26	352
	156.3	8.19	345
	312.5	8.14	347
	Cycl.	8.20	346

EMS: Ethyl methanesulphonate(1 µL/mL)

Cycl.: Cyclophosphamide (5.0 µg/mL)

Table 14: pH AND Osmolality Data, Experiment B

 

Concentration (µg/mL)		pH	Osmolality (mmol/kg)
20-hour treatment without S9 Mix / 20-hour and 28-hor sampling times
Negative (solvent) Control		8.16	347
Solubilised Sulphur Black 1	9.8	8.35	345
	19.6	8.37	342
	39.1	8.42	350
	58.7	8.40	347
	EMS	8.36	352
3-hour treatment with S9 Mix / 28-hour sampling time
Negative (solvent) Control		8.20	347
Solubilised Sulphur Black 1	39.1	8.26	350
	78.2	8.22	352
	156.3	8.25	344
	312.5	8.18	347
	Cycl.	8.17	344

EMS:Ethyl methanesulphonate(0.4 µL/mL )

Cycl.:Cyclophosphamide (5.0 µg/mL)

Conclusions:

In an in vitro chromosome aberration assay (CA) according to OECD guideline 473, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

Executive summary:

The clastogenic potential of the test item was tested in an in vitro chromosome aberration (CA) assay according to OECD guideline 473 in V79 (Chinese hamster lung cellline) in two independent experiments.For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix)

Experiment A with 3/20 h treatment/sampling time

without S9 mix:                39.1, 78.2, 156.3 and 312.5 µg/mL test item

with S9 mix:                   39.1, 78.2, 156.3 and 312.5 µg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix:              9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix:              9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix:                   39.1, 78.2, 156.3 and 312.5 µg/mL test item

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 (Giemsa). In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

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 presence of metabolic activation, three 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 relationship was 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 µL/mL) (without S9 mix) or cyclophosphamide (5.0 µg/mL) (with S9 mix) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were comparable with the historical positive control data. Thus, the study is considered valid.

In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 October 2017- 03 January 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:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian gene mutation assay using the Hprt gene.

Specific details on test material used for the study:

Expiration date: 30 Nov 2021

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) located on the X chromosome.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

K1 subline

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- Methods for maintenance in cell culture: The cell stocks are kept in liquid nitrogen. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks. The CHO K1 cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).

MEDIA USED
- Type and identity of media including CO2 concentration: Ham´s F12 Medium, 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of phenobarbital and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

Mutation Assay 5-hour treatment period without S9-mix:
1250, 2500, 2750, 3000, 3500, and 4000 µg/mL

Mutation Assay 5-hour treatment period with S9-mix:
312.5, 625, 1250, 2500, and 5000 µg/mL

The results of the pre-test on cell toxicity were used for dose selection of the test item concentrations used in the main mutation assay.

Vehicle / solvent:

- Solvent used: Ham's F12 medium
- Justification for choice of solvent: This solvent was chosen based on the results of the preliminary solubility test and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without S9 mix, 1.0 µL/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With S9 mix, 20 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
- Cell density at seeding: 10E6 cells/dish

DURATION
- Preincubation period: 24 h
- Exposure duration: 5 h
- Expression time (cells in growth medium): 8 d

SELECTION AGENT (mutation assays): thioguanine (6-TG)

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The colonies were fixed with methanol for five minutes, stained with Giemsa

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Evaluation of Results
Providing that all acceptability criteria are fulfilled, the test item is considered to be clearly positive if, in any of the experimental conditions examined:
•at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
•any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit)
•the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
•none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
•there is no concentration-related increase when evaluated with an appropriate trend test
•all results are inside the distribution of the historical negative control data (based 95% control limit)
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:
•mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups
•mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control
ite treated groups
•The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH or osmolality: The osmolality and pH values of test item solutions did not show any relevant alterations when compared to the concurrent control groups in the pre-test
- Precipitation: There was no precipitation of the test item at any dose level tested.

RANGE-FINDING/SCREENING STUDIES:
A study for cytotoxicity and solubility was performed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (95%)
- Positive historical control data:
Without S9 mix (EMS)
Mean: 1517.44; SD 29.86; Lower confidence interval 1455.15; upper confidence interval 1579.73
With S9 mix (DMBA)
Mean: 745.11; SD 16.86; Lower confidence interval 709.95; upper confidence interval 780.27

- Solvent historical control data:
Without S9 mix
Mean: 6.72; SD .066; Lower confidence interval 5.35; upper confidence interval 8.10
With S9 mix
Mean: 6.89; SD 0.81; Lower confidence interval 5.20; upper confidence interval 8.58

Summarized Results of the PRE-TEST ON TOXICITY (CONCENTRATION SELECTION)

Table 1: 5-hour treatment with and without S9-mix

Test group	Dose µg/mL	S9-mix	Treatment/ time/ hour	Number of colonies/200cells/dish			Mean	Relative^a survival in percent
				dish 1	dish 2	dish 3
Solvent Control (Ham's F12 medium)	–	–	5	205	206	201	204,0	100
Solubilised Sulphur Black 1	39.1	–	5	203	202	206	203,7	100
	78.2	–	5	200	201	199	200,0	98
	156.3	–	5	204	204	200	202,7	99
	312.5	–	5	203	204	205	204,0	100
	625	–	5	200	199	201	200,0	98
	1250	–	5	198	200	197	198,3	97
	2500	–	5	160	162	158	160,0	78
	5000	–	5	0	0	0	0,0	0
Solvent Control Ham's F12 medium)	–	+	5	200	204	204	202,7	100
Solubilised Sulphur Black 1	39.1	+	5	205	202	199	202,0	100
	78.2	+	5	203	201	203	202,3	100
	156.3	+	5	197	202	201	200,0	99
	312.5	+	5	201	201	205	202,3	100
	625	+	5	198	199	203	200,0	99
	1250	+	5	204	202	200	202,0	100
	2500	+	5	198	203	199	200,0	99
	5000	+	5	201	197	202	200,0	99

^a Relative to Solvent Control

 

CHO/HPRT MUTAGENESIS ASSAY RESULTS, MAIN MUTATION ASSAY/a, b, c and d

Table 2: 5-hour Treatment without S9-Mix 

Study code:				805-476-2715
Test item:				Solubilised Sulphur Black 1 (without S9-mix)				Batch number:						2401003784
Test date of Main Mutation Assay:				October 10, 2017 - October 26, 2017				Expression period:						8 days
Solvent:				Ham's F12 medium				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control a	201.3	±	2.31		100	100	1		2	3	1	1	8		101	7.92
Pos. control (EMS 1.0µL/mL) a	53.3	±	2.08		26	71	198		203	200	195	207	1003		72	1393.06**
TEST ITEM
1250g/mL a	201,7	±	1,53		100	100	1		0	3	3	0	7		100	7.00
2500g/mL a	161,3	±	1,53		80	101	0		2	2	1	1	6		102	5.88
2750g/mL a	151,0	±	1,00		75	100	2		2	1	2	0	7		101	6.93
3000g/mL a	127,3	±	1,15		63	99	1		3	1	1	3	9		100	9.00
3500g/mL a	36,3		2,08		18	99	1		3	2	3	2	11		100	11.00*

a = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

EMS=Ethyl methanesulfonate

* = p < 0.05 to the historical control

** = p < 0.01 to the concurrent negative control and to the historical control

 

 

Table 3: 5-hour Treatment without S9-Mix 

Study code:				805-476-2715
Test item:				Solubilised Sulphur Black 1 (without S9-mix)				Batch number:						2401003784
Test date of Main Mutation Assay:				October 10, 2017 - October 26, 2017				Expression period:						8 days
Solvent:				Ham's F12 medium				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control b	202.7	±	0.58		100	100	1		1	2	2	1	7		101	6.93
Pos. control (EMS 1.0µL/mL) b	53.0	±	2.65		26	71	210		205	195	194	199	1003		72	1393.06**
TEST ITEM
1250g/mL b	199,7	±	0,58		99	99	1		3	0	2	1	7		101	6.93
2500g/mL b	160,0	±	1,00		79	100	1		2	1	2	0	6		101	5.94
2750g/mL b	151,0	±	1,00		75	99	2		2	2	0	0	6		100	6.00
3000g/mL b	129,0	±	1,00		64	100	3		1	1	2	1	8		101	7.92
3500g/mL b	34,0	±	1,00		17	99	3		3	0	2	2	10		100	10.00

b = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

EMS=Ethyl methanesulfonate

** = p < 0.01 to the concurrent negative control and to the historical control

 Table 4: 5-hour Treatment without S9-Mix 

Study code:				805-476-2715
Test item:				Solubilised Sulphur Black 1 (without S9-mix)				Batch number:						2401003784
Test date of Main Mutation Assay:				October 10, 2017 - October 26, 2017				Expression period:						8 days
Solvent:				Ham's F12 medium				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control c	202.0	±	3.46		100	100	2		1	1	1	2	7		101	6.93
Pos. control (EMS 1.0µL/mL) c	55.0	±	1.73		28	66	207		202	192	197	206	1004		67	1498.51**
TEST ITEM
1250g/mL c	199,0	±	2,00		99	99	0		3	0	3	1	7		100	7.00
2500g/mL c	162,3	±	2,08		80	98	1		1	2	2	1	7		99	7.07
2750g/mL c	154,0	±	1,00		76	98	1		1	3	0	2	7		99	7.07
3000g/mL c	127,3	±	2,52		63	99	4		1	1	3	0	9		100	9.00
3500g/mL c	37,0	±	1,00		18	99	4		0	2	1	3	10		100	10.00

c = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

EMS=Ethyl methanesulfonate

** = p < 0.01 to the concurrent negative control and to the historical control

 

Table 5: 5-hour Treatment without S9-Mix

Study code:				805-476-2715
Test item:				Solubilised Sulphur Black 1 (without S9-mix)				Batch number:						2401003784
Test date of Main Mutation Assay:				October 10, 2017 - October 26, 2017				Expression period:						8 days
Solvent:				Ham's F12 medium				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control d	202.0	±	2.00		100	100	1		2	2	1	2	8		101	7.92
Pos. control (EMS 1.0µL/mL) d	54.0	±	1.00		27	67	208		204	197	193	198	1000		68	1470.59**
TEST ITEM
1250g/mL d	198,0	±	1,73		98	99	0		3	2	1	0	6		100	6.00
2500g/mL d	161,0	±	1,73		80	99	1		1	2	3	0	7		100	7.00
2750g/mL d	154,0	±	0,00		76	99	1		2	0	2	2	7		100	7.00
3000g/mL d	127,0	±	2,00		63	100	1		1	2	3	2	9		100	9.00
3500g/mL d	37,3	±	2,08		18	99	3		3	2	2	2	12		100	12.00^##

d = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

EMS=Ethyl methanesulfonate

** = p < 0.01 to the concurrent negative control and to the historical control

##== p < 0.01 to the historical control

 

Table 6: 5-hour Treatment with S9 -Mix 

Study code:				805-476-2715
Test item:				Solubilised Sulphur Black 1 (with S9-mix)				Batch number:						2401003784
Test date of Main Mutation Assay:				October 10, 2017 - October 26, 2017				Expression period:						8 days
Solvent:				Ham's F12 medium				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control a	202.0	±	2.00		100	100	2		1	1	1	1	6		100	6.00
Pos. control (DMBA20 µg/mL) a	125.0	±	1.00		62	77	112		110	117	108	105	552		77	716.88**
TEST ITEM
312.5g/mL a	201,7	±	2,52		100	99	0		2	2	2	0	6		99	6.06
625g/mL a	202,3	±	2,08		100	99	1		2	3	0	1	7		99	7.07
1250g/mL a	203,3	±	2,52		101	98	0		1	2	1	2	6		98	6.12
2500g/mL a	198,7	±	1,15		98	99	2		0	2	1	2	7		99	7.07
5000g/mL a	199,0	±	2,00		99	98	1		3	2	0	1	7		98	7.14

a = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

DMBA=7,12-Dimethyl benzanthracene

** = p < 0.01 to the concurrent negative control and to the historical control

 

 

Table 7: 5-hour Treatment with S9 -Mix  

Study code:				805-476-2715
Test item:				Solubilised Sulphur Black 1 (with S9-mix)				Batch number:						2401003784
Test date of Main Mutation Assay:				October 10, 2017 - October 26, 2017				Expression period:						8 days
Solvent:				Ham's F12 medium				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control b	201.7	±	1.53		100	100	2		0	1	1	3	7		100	7.00
Pos. control (DMBA20 µg/mL) b	125.3	±	0.58		62	76	119		111	109	114	106	559		76	735.53**
TEST ITEM
312.5g/mL b	199,7	±	0,58		99	99	0		3	0	1	3	7		99	7.07
625g/mL b	201,3	±	1,53		100	99	1		1	2	1	2	7		99	7.07
1250g/mL b	201,0	±	1,00		100	99	2		0	1	1	2	6		99	6.06
2500g/mL b	198,7	±	1,15		99	99	2		2	1	1	1	7		100	7.00
5000g/mL b	199,7	±	1,53		99	99	0		3	1	0	3	7		99	7.07

b = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

DMBA=7,12-Dimethyl benzanthracene

** = p < 0.01 to the concurrent negative control and to the historical control

 

 

 

Table 8: 5-hour Treatment with S9 -Mix 

Study code:				805-476-2715
Test item:				Solubilised Sulphur Black 1 (with S9-mix)				Batch number:						2401003784
Test date of Main Mutation Assay:				October 10, 2017 - October 26, 2017				Expression period:						8 days
Solvent:				Ham's F12 medium				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control c	200.7	±	2.31		100	100	1		3	0	2	0	6		100	6.00
Pos. control (DMBA20 µg/mL) c	126.3	±	3.21		63	70	107		114	106	102	110	539		70	770.00**
TEST ITEM
312.5g/mL c	203,3	±	1,53		101	99	3		3	0	0	0	6		99	6.06
625g/mL c	201,0	±	2,00		100	100	1		1	2	2	1	7		100	7.00
1250g/mL c	199,3	±	1,53		99	98	1		2	0	2	2	7		98	7.14
2500g/mL c	201,3	±	1,53		100	98	1		2	2	1	0	6		98	6.12
5000g/mL c	200,0	±	2,00		100	99	1		1	1	3	0	6		99	6.06

c = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

DMBA=7,12-Dimethyl benzanthracene

** = p < 0.01 to the concurrent negative control and to the historical control

 

 

Table 9: 5-hour Treatment with S9 -Mix 

Study code:				805-476-2715
Test item:				Solubilised Sulphur Black 1 (with S9-mix)				Batch number:						2401003784
Test date of Main Mutation Assay:				October 10, 2017 - October 26, 2017				Expression period:						8 days
Solvent:				Ham's F12 medium				Selective agent:						3.4 µg/mL 6-thioguanine
Cells seeded for analysis:				2x10^5cells /dish for mutant selection: 200 cells/dish for C.E.
NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT					REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER						TOTAL MUTANT COLONIES		ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.				PERCENT VEH. CONTROL		1		2	3	4	5
Solvent control d	202.3	±	0.58		100	100	1		1	1	2	1	6		100	6.00
Pos. control (DMBA20 µg/mL) d	124.3	±	2.31		61	70	115		108	105	109	101	538		70	768.57**
TEST ITEM
312.5g/mL d	201,0	±	1,00		99	99	1		2	2	1	1	7		99	7.07
625g/mL d	200,7	±	2,08		99	99	3		0	1	2	1	7		100	7.00
1250g/mL d	200,0	±	1,73		99	98	2		1	2	0	2	7		98	7.14
2500g/mL d	201,3	±	0,58		100	99	1		2	1	1	1	6		99	6.06
5000g/mL d	199,3	±	1,15		99	99	1		1	2	3	0	7		99	7.07

d = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

DMBA=7,12-Dimethyl benzanthracene

** = p < 0.01 to the concurrent negative control and to the historical control

Table 10: Day 1 Cloning efficiencies -Main Mutation Assay (5-hour treatment without And with S9-Mix) 

Test Group a and b	Concentration µg/mL	S9-mix	Treatment time/ hour	Number of colonies/200cells/dish				Cloning efficiency (%)	% of Control
				dish 1	dish 2	dish 3	Mean
Negative (Solvent Control)	-	–	5	204	200	200	201,3	101	100
Solubilised Sulphur Black 1- Group a	1250	–	5	200	202	203	201,7	101	100
	2500	–	5	160	161	163	161,3	81	80
	2750	–	5	150	151	152	151,0	76	75
	3000	–	5	126	128	128	127,3	64	63
	3500	–	5	38	34	37	36,3	18	18
	4000	–	5	0	0	0	0,0	0	0
	EMS (1µL/mL)	–	5	54	51	55	53,3	27	26
Negative (Solvent Control)	-	–	5	203	202	203	202,7	101	100
Solubilised Sulphur Black 1 - Group b	1250	–	5	199	200	200	199,7	100	99
	2500	–	5	159	161	160	160,0	80	79
	2750	–	5	151	150	152	151,0	76	75
	3000	–	5	130	129	128	129,0	65	64
	3500	–	5	33	34	35	34,0	17	17
	4000	–	5	0	0	0	0,0	0	0
	EMS (1µL/mL)	–	5	55	50	54	53,0	27	26

EMS= Ethyl methanesulfonate

Table 11: Day 1 Cloning efficiencies -Main Mutation Assay (5-hour treatment without And with S9-Mix) 

Test Group c and d	Concentration µg/mL	S9-mix	Treatment time/ hour	Number of colonies/200cells/dish				Cloning efficiency (%)	% of Control
				dish 1	dish 2	dish 3	Mean
Negative (Solvent Control)	-	–	5	198	204	204	202,0	101	100
Solubilised Sulphur Black 1- Group c	1250	–	5	197	199	201	199,0	100	99
	2500	–	5	164	160	163	162,3	81	80
	2750	–	5	155	154	153	154,0	77	76
	3000	–	5	130	127	125	127,3	64	63
	3500	–	5	38	37	36	37,0	19	18
	4000	–	5	0	0	0	0,0	0	0
	EMS (1µL/mL)	–	5	53	56	56	55,0	28	27
Negative (Solvent Control)	-	–	5	204	200	202	202,0	101	100
Solubilised Sulphur Black 1 - Group d	1250	–	5	197	197	200	198,0	99	98
	2500	–	5	160	160	163	161,0	81	80
	2750	–	5	154	154	154	154,0	77	76
	3000	–	5	129	127	125	127,0	64	63
	3500	–	5	39	38	35	37,3	19	18
	4000	–	5	0	0	0	0,0	0	0
	EMS (1µL/mL)	–	5	55	54	53	54,0	27	27

EMS= Ethyl methanesulfonate

Table 12: Day 1 Cloning efficiencies -Main Mutation Assay (5-hour treatment without And with S9-Mix) 

Test group a and b	Concentration µg/mL	S9-mix	Treatment time/ hour	Number of colonies/200cells/dish				Cloning efficiency (%)	% of Control
				dish 1	dish 2	dish 3	Mean
Negative (Solvent Control)	-	+	5	200	204	202	202,0	101	100
Solubilised Sulphur Black 1- Group a	312.5	+	5	204	202	199	201,7	101	100
	625	+	5	203	204	200	202,3	101	100
	1250	+	5	206	203	201	203,3	102	101
	2500	+	5	200	198	198	198,7	99	98
	5000	+	5	199	201	197	199,0	100	99
	DMBA (20µg/mL)	+	5	125	124	126	125,0	63	62
Negative (Solvent Control)	-	+	5	200	202	203	201,7	101	100
Solubilised Sulphur Black 1-Groupb	312.5	+	5	200	200	199	199,7	100	99
	625	+	5	201	203	200	201,3	101	100
	1250	+	5	202	200	201	201,0	101	100
	2500	+	5	200	198	198	198,7	99	99
	5000	+	5	200	201	198	199,7	100	99
	DMBA (20µg/mL)	+	5	125	125	126	125,3	63	62

DMBA= 7,12-Dimethylbenzanthracene

Table 13: Day 1 Cloning efficiencies -Main Mutation Assay (5-hour treatment without And with S9-Mix) 

Test group c and d	Concentration µg/mL	S9-mix	Treatment time/ hour	Number of colonies/200cells/dish				Cloning efficiency (%)	% of Control
				dish 1	dish 2	dish 3	Mean
Negative (Solvent Control)	-	+	5	202	198	202	200,7	100	100
Solubilised Sulphur Black 1- Group c	312.5	+	5	203	202	205	203,3	102	101
	625	+	5	199	201	203	201,0	101	100
	1250	+	5	198	201	199	199,3	100	99
	2500	+	5	200	201	203	201,3	101	100
	5000	+	5	202	200	198	200,0	100	100
	DMBA (20µg/mL)	+	5	124	125	130	126,3	63	63
Negative (Solvent Control)	-	+	5	202	203	202	202,3	101	100
Solubilised Sulphur Black 1-Groupd	312.5	+	5	201	200	202	201,0	101	99
	625	+	5	200	199	203	200,7	100	99
	1250	+	5	199	202	199	200,0	100	99
	2500	+	5	201	201	202	201,3	101	100
	5000	+	5	200	198	200	199,3	100	99
	DMBA (20µg/mL)	+	5	123	123	127	124,3	62	61

DMBA= 7,12-Dimethylbenzanthracene

Table 14: Day 8 Cloning efficiencies -Main Mutation Assay (5-hour treatment without and with S9-Mix) 

Test Group a and b	Concentration µg/mL	S9-mix	Treatment time/ hour	Number of colonies/200cells/dish				Cloning efficiency (%)	% of Control
				dish 1	dish 2	dish 3	Mean
Negative (Solvent Control)	-	–	5	202	200	203	201,7	101	100
Solubilised Sulphur Black 1- Group a	1250	–	5	203	200	199	200,7	100	100
	2500	–	5	202	204	204	203,3	102	101
	2750	–	5	202	199	202	201,0	101	100
	3000	–	5	199	198	203	200,0	100	99
	3500	–	5	197	201	199	199,0	100	99
	4000	–	5	0	0	0	0,0	0	0
	EMS (1µL/mL)	–	5	142	147	142	143,7	72	71
Negative (Solvent Control)	-	–	5	200	204	203	202,3	101	100
Solubilised Sulphur Black 1 - Group b	1250	–	5	201	202	200	201,0	101	99
	2500	–	5	200	203	202	201,7	101	100
	2750	–	5	200	200	201	200,3	100	99
	3000	–	5	201	200	203	201,3	101	100
	3500	–	5	198	200	200	199,3	100	99
	4000	–	5	0	0	0	0,0	0	0
	EMS (1µL/mL)	–	5	143	144	143	143,3	72	71

EMS= Ethyl methanesulfonate

Table 15: Day 8 Cloning efficiencies -Main Mutation Assay (5-hour treatment without and with S9-Mix)

Test Group c and d	Concentration µg/mL	S9-mix	Treatment time/ hour	Number of colonies/200cells/dish				Cloning efficiency (%)	% of Control
				dish 1	dish 2	dish 3	Mean
Negative (Solvent Control)	-	–	5	203	202	203	202,7	101	100
Solubilised Sulphur Black 1- Group c	1250	–	5	203	198	198	199,7	100	99
	2500	–	5	196	198	202	198,7	99	98
	2750	–	5	197	195	203	198,3	99	98
	3000	–	5	200	197	202	199,7	100	99
	3500	–	5	200	200	200	200,0	100	99
	4000	–	5	0	0	0	0,0	0	0
	EMS (1µL/mL)	–	5	120	122	117	119,7	60	59
Negative (Solvent Control)	-	–	5	201	202	202	201,7	101	100
Solubilised Sulphur Black 1 - Group d	1250	–	5	202	200	199	200,3	100	99
	2500	–	5	199	200	202	200,3	100	99
	2750	–	5	198	199	201	199,3	100	99
	3000	–	5	199	201	202	200,7	100	100
	3500	–	5	199	199	200	199,3	100	99
	4000	–	5	0	0	0	0,0	0	0
	EMS (1µL/mL)	–	5	119	121	118	119,3	60	59

EMS= Ethyl methanesulfonate

 

 

Table 16: Day 8 Cloning efficiencies -Main Mutation Assay (5-hour treatment without and with S9-Mix)

Test group a and b	Concentration µg/mL	S9-mix	Treatment time/ hour	Number of colonies/200cells/dish				Cloning efficiency (%)	% of Control
				dish 1	dish 2	dish 3	Mean
Negative (Solvent Control)	-	+	5	200	202	197	199,7	100	100
Solubilised Sulphur Black 1- Group a	312.5	+	5	198	199	197	198,0	99	99
	625	+	5	195	200	198	197,7	99	99
	1250	+	5	197	194	199	196,7	98	98
	2500	+	5	195	199	197	197,0	99	99
	5000	+	5	195	194	196	195,0	98	98
	DMBA (20µg/mL)	+	5	155	151	157	154,3	77	77
Negative (Solvent Control)	-	+	5	199	202	201	200,7	100	100
Solubilised Sulphur Black 1-Groupb	312.5	+	5	199	199	198	198,7	99	99
	625	+	5	196	199	198	197,7	99	99
	1250	+	5	198	197	199	198,0	99	99
	2500	+	5	198	199	200	199,0	100	99
	5000	+	5	197	198	200	198,3	99	99
	DMBA (20µg/mL)	+	5	151	152	155	152,7	76	76

DMBA= 7,12-Dimethylbenzanthracene

 

Table 17: Day 8 Cloning efficiencies -Main Mutation Assay (5-hour treatment without and with S9-Mix)

Test group c and d	Concentration µg/mL	S9-mix	Treatment time/ hour	Number of colonies/200cells/dish				Cloning efficiency (%)	% of Control
				dish 1	dish 2	dish 3	Mean
Negative (Solvent Control)	-	+	5	200	201	199	200,0	100	100
Solubilised Sulphur Black 1- Group c	312.5	+	5	195	199	200	198,0	99	99
	625	+	5	197	200	201	199,3	100	100
	1250	+	5	194	197	195	195,3	98	98
	2500	+	5	195	199	191	195,0	98	98
	5000	+	5	198	196	197	197,0	99	99
	DMBA (20µg/mL)	+	5	140	141	138	139,7	70	70
Negative (Solvent Control)	-	+	5	202	200	199	200,3	100	100
Solubilised Sulphur Black 1-Groupd	312.5	+	5	197	199	199	198,3	99	99
	625	+	5	198	198	201	199,0	100	99
	1250	+	5	195	197	197	196,3	98	98
	2500	+	5	199	200	195	198,0	99	99
	5000	+	5	197	199	197	197,7	99	99
	DMBA (20µg/mL)	+	5	139	141	140	140,0	70	70

DMBA= 7,12-Dimethylbenzanthracene

 

Table 18: pH and Osmolality Data in Pre-test on Toxicity (Concentration selection)

Concentration (µg/mL)		pH	Osmolality (mmol/kg)
Treatment period (hours): 5/without S9-Mix
Negative (solvent) Control		7.68	292
Solubilised Sulphur Black 1	39.1	7.63	297
	78.2	7.61	297
	156.3	7.60	296
	312.5	7.64	299
	625	7.65	302
	1250	7.66	316
	2500	7.76	321
	5000	7.84	330
Treatment period (hours): 5/with S9-Mix
Negative (solvent) Control		7.31	300
Solubilised Sulphur Black 1	39.1	7.29	300
	78.2	7.28	299
	156.3	7.32	299
	312.5	7.34	301
	625	7.39	305
	1250	7.48	312
	2500	7.55	319
	5000	7.73	330

 

Table 19: pH and Osmolality Data in Main Mutation Assay

Concentration (µg/mL)			pH		Osmolality (mmol/kg)
Treatment period (hours): 5/without S9-Mix
Negative (solvent) Control			7.58		309
Solubilised Sulphur Black 1	1250		7.77		307
	2500		7.79		311
	2750		7.78		314
	3000		7.70		316
	3500		7.79		317
	4000		7.71		321
EMS	1.00 µL/mL		7.71		299
Treatment period (hours): 5/with S9-Mix
Negative (solvent) Control		7.42		301
Solubilised Sulphur Black 1	312.5		7.48		304
	625		7.48		305
	1250		7.52		304
	2500		7.56		306
	5000		7.63		311
DMBA	20 µg/mL		7.45		558

 

EMS= Ethyl methanesulfonate,

DMBA= 7,12-Dimethylbenzanthracene

Conclusions:

In an in vitro mammalian gene mutation test (HPRT) in CHO-K1 cells according to OECD guideline 476, the test item did not show mutagenic properties.

Executive summary:

The mutagenic potential of the test item was tested in an 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:

Mutation Assay: 5-hour treatment period without S9-mix:

1250, 2500, 2750, 3000, 3500, and 4000 µg/mL 

Mutation Assay: 5-hour treatment period with S9-mix:

312.5, 625, 1250, 2500 and 5000 µg/mL

Phenotypic expression was evaluated up to 8 days following exposure. There was no precipitation of the test item observable at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. In the absence of metabolic activation clear cytotoxicity (survival ca. 20%) of the test item was observed at 4000 µg/mL, whereas in the presence of metabolic activation, no cytotoxicity was observed up to the highest tested concentration of 5000 µg/mL.

The mutation frequency found in the solvent controls was in the range of the 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 when compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control at any concentration tested in the absence and presence of metabolic activation. In the presence of S9 mix, there were no statistically significant differences to the historical control data and no dose-dependency was observed. In the absence of S9 mix, in the cultures treated with 3000 and 3500 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (3 of 4 and 4 of 4 cultures, respectively). Statistically significant differences to the historical control data were observed at concentration 3500 µg/mL in 2 of 4 cultures. These findings were considered not 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 were observed. 

The test item was tested up to the maximum recommended concentration with and without metabolic activation over a 5 hour treatment period did not induce biologically relevant increases in mutant frequency. 

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test under the given conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (Ames) 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 S9 mix prepared from livers of phenobarbital/beta-naphthoflavone-induced rats. The study included preliminary solubility test, preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was solved in ultrapure water( ASTM Type I). At the formulation of test item solutions correction of concentrations for active component content (80.2 % dyestuff) was made in the main 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, and 16 µg/plate. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study. The test item did not show unequivocal inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) were considered to be within the biological variability range of the applied test system. The revertant colony numbers of solvent control (ultrapure water (ASTM Type I)) 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, 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 with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

The clastogenic potential of the test item was tested in an in vitro chromosome aberration (CA) assay according to OECD guideline 473 in V79 (Chinese hamster lung cellline) in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test (without and with metabolic activation using rodent S9 mix).

Experiment A with 3/20 h treatment/sampling time

without S9 mix:                39.1, 78.2, 156.3 and 312.5 µg/mL test item

with S9 mix:                   39.1, 78.2, 156.3 and 312.5 µg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix:              9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix:              9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix:                   39.1, 78.2, 156.3 and 312.5 µg/mL test item

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 (Giemsa). In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. 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 presence of metabolic activation, three 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 relationship was 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 µL/mL) (without S9 mix) or cyclophosphamide (5.0 µg/mL) (with S9 mix) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were comparable with the historical positive control data. Thus, the study is considered valid.

In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

The mutagenic potential of the test item was tested in an 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:

Mutation Assay: 5-hour treatment period without S9-mix:

1250, 2500, 2750, 3000, 3500, and 4000 µg/mL 

Mutation Assay: 5-hour treatment period with S9-mix:

312.5, 625, 1250, 2500 and 5000 µg/mL

Phenotypic expression was evaluated up to 8 days following exposure. There was no precipitation of the test item observable at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. In the absence of metabolic activation clear cytotoxicity (survival ca.20%) of the test item was observed at 4000 µg/mL, whereas in the presence of metabolic activation, no cytotoxicity was observed up to the highest tested concentration of 5000 µg/mL.

The mutation frequency found in the solvent controls was in the range of the 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 when compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control at any concentration tested in the absence and presence of metabolic activation. In the presence of S9 mix, there were no statistically significant differences to the historical control data and no dose-dependency was observed. In the absence of S9 mix, in the cultures treated with 3000 and 3500 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (3 of 4 and 4 of 4 cultures, respectively). Statistically significant differences to the historical control data were observed at concentration 3500 µg/mL in 2 of 4 cultures. 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 were observed. 

The test item was tested up to the maximum recommended concentration with and without metabolic activation over a 5 hour treatment period and did not induce biologically relevant increases in mutant frequency. 

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test under the given conditions.

The test item did not induce gene mutation in a bacterial or mammalian test system. Furthermore, the test item did not induce chromosome aberration in another mammalian test system. According to these results, the test item is considered to be non-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. As a result the test substance is considered not to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.