Reaction product of 4-aminophenol with 2-ethyl-6-methylbenzenamine and sodium polysulfide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

Based on the results of an OECD 476 study, the test substance is considered to be non-mutagenic in Chinese hamster ovary cells.

Based on the results of an OECD 473 study, the test substance is considered to be non-clastogenic in Chinese Hamster lung V79 cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2017 - 12 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

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)

Test concentrations with justification for top dose:

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

Vehicle / solvent:

dimethyl sulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

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

Details on test system and experimental conditions:

Origin of the Bacterial Strains

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

Storage of Tester Strains

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

Confirmation of Phenotypes of Tester Strains

The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al..

Spontaneous Reversion of Tester Strains

Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.

Procedure for Bacterial Cultures

The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 (Section: 5.4.2) for the overnight cultures in the assay. The cultures were incubated for approximately 11-14 hours in a 37oC Benchtop Incubator Shaker.

Viability and the Cell Count of the Testing Bacterial Cultures

The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Metabolic Activation System

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

Rat Liver S9 Fraction

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

Rationale for test conditions:

Justification of concentrations:
Selection of the concentration range was done on the basis of solubility tests and concentration range finding tests (informatory toxicity tests). In the solubility tests the test item behavior was investigated in the applied test system when formulated in ultrapure water or DMSO. In the preliminary phase of this study two pre-experiments were performed to find the most appropriate solvent for the main experiments.

Based on the solubility tests, stock suspensions with a concentration of 50 mg/mL were prepared in ultrapure water and 25 mg/mL in dimethyl sulfoxide (DMSO), respectively and diluted accordingly. In the informatory toxicity tests any correction factor, based on the active component of the test item (87 %) was not taken into consideration; therefore the 50 and 25 mg/mL stock suspension concentrations corresponded to 43.5 and 21.8 mg active component/mL. 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 in both tests.

Evaluation criteria:

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

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle 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 vehicle control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

not examined

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:

not examined

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:

not examined

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:

not examined

Positive controls validity:

valid

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	24.0	1.57	26.7	1.51	96.3	1.11	101.3	1.06	12.7	1.27	11.0	1.10	7.0	0.91	8.0	1.14	22.7	0.89	32.3	1.31
DMSO Control (100 µL)	15.3	1.00	21.3	1.00	87.0	1.00	95.0	1.00	11.7	1.00	12.3	1.00	7.0	1.00	8.3	1.00	22.0	1.00	37.0	1.00
DMSO Control (200µL)	15.3	1.00	17.7	1.00	86.7	1.00	95.7	1.00	10.0	1.00	10.0	1.00	7.7	1.00	7.0	1.00	25.3	1.00	24.7	1.00
Ultrapure Water Control	–	–	–	–	85.3	1.00	–	–	9.7	1.00	–	–	–	–	–	–	25.0	1.00	–	–
5000	10.3	0.67	13.3	0.75	85.0	0.98	78.7	0.82	7.7	0.77	9.3	0.93	0.0	0.00	0.3	0.05	21.0	0.83	27.3	1.11
1600	11.7	0.76	18.0	1.02	79.0	0.91	100.3	1.05	7.7	0.77	9.3	0.93	0.0	0.00	12.7	1.81	21.3	0.84	25.0	1.01
500	19.3	1.26	21.0	1.19	98.0	1.13	94.7	0.99	10.3	1.03	13.0	1.30	5.7	0.74	20.7	2.95	23.0	0.91	24.0	0.97
160	16.7	1.09	27.0	1.53	86.3	1.00	104.7	1.09	9.7	0.97	9.7	0.97	9.0	1.17	12.3	1.76	23.0	0.91	19.3	0.78
50	10.7	0.70	23.3	1.32	94.7	1.09	86.3	0.90	11.3	1.13	10.7	1.07	7.3	0.96	13.0	1.86	19.7	0.78	26.0	1.05
16	17.3	1.13	27.7	1.57	87.0	1.00	91.0	0.95	7.7	0.77	6.3	0.63	9.0	1.17	6.7	0.95	22.7	0.89	25.7	1.04
5	20.7	1.35	27.0	1.53	82.7	0.95	100.7	1.05	8.7	0.87	11.7	1.17	8.7	1.13	7.3	1.05	21.3	0.84	30.0	1.22
NPD (4mg)	240.3	15.67	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1154.7	13.53	–	–	962.7	99.59	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	318.0	45.43	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	644.0	25.76	–	–
2AA (2mg)	–	–	1325.3	62.13	–	–	1429.3	15.05	–	–	146.7	11.89	–	–	95.7	11.48	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	204.0	5.51

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

Remarks:           DMSO (200 µL) was applied as solvent of the test item and DMSO (100 µL) was applied as solvent of the positive control substances 9AA, NPD and 2AA. The ultrapure water (100 µL) was applied as solvent of the positive control substances MMS and SAZ. The mutation rate of the test item and the untreated control refers to the DMSO sample (200 µL); the mutation rate of the 9AA, NPD and 2AA refers to the DMSO sample (100 µL). The mutation rate of MMS and SAZ refers to ultrapure water (100 µL).

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				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	16.7	1.14	24.0	1.16	99.3	1.27	115.0	1.58	9.3	1.04	9.7	1.07	8.0	1.33	9.0	1.08	34.3	1.37	37.3	1.12
DMSO Control (100 µL)	21.7	1.00	18.0	1.00	98.7	1.00	90.7	1.00	9.3	1.00	10.0	1.00	7.3	1.00	7.7	1.00	27.7	1.00	35.7	1.00
DMSO Control (200µL)	14.7	1.00	20.7	1.00	78.3	1.00	73.0	1.00	9.0	1.00	9.0	1.00	6.0	1.00	8.3	1.00	25.0	1.00	33.3	1.00
Ultrapure Water Control	–	–	–	–	100.7	1.00	–	–	11.3	1.00	–	–	–	–	–	–	29.7	1.00	–	–
5000	4.3	0.30	11.7	0.56	25.0	0.32	53.7	0.74	3.3	0.37	7.0	0.78	0.0	0.00	4.3	0.52	25.0	1.00	32.7	0.98
1600	7.3	0.50	15.3	0.74	45.3	0.58	103.0	1.41	5.7	0.63	5.7	0.63	0.0	0.00	4.3	0.52	28.7	1.15	27.7	0.83
500	9.0	0.61	17.3	0.84	72.7	0.93	106.7	1.46	7.3	0.81	11.3	1.26	1.3	0.22	16.7	2.00	34.7	1.39	30.0	0.90
160	16.7	1.14	20.7	1.00	79.7	1.02	84.7	1.16	10.0	1.11	8.0	0.89	6.7	1.11	15.7	1.88	32.7	1.31	31.0	0.93
50	13.7	0.93	20.3	0.98	100.3	1.28	93.3	1.28	7.7	0.85	10.0	1.11	5.0	0.83	18.0	2.16	23.7	0.95	38.7	1.16
16	14.0	0.95	23.3	1.13	83.3	1.06	87.7	1.20	9.3	1.04	10.0	1.11	7.7	1.28	12.7	1.52	20.3	0.81	32.0	0.96
5	10.7	0.73	23.3	1.13	77.3	0.99	86.0	1.18	9.7	1.07	12.3	1.37	8.7	1.44	6.7	0.80	21.0	0.84	31.7	0.95
NPD (4mg)	226.0	10.43	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1829.3	18.17	–	–	525.3	46.35	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	273.7	37.32	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1344.0	45.30	–	–
2AA (2mg)	–	–	1213.3	67.41	–	–	1626.7	17.94	–	–	148.3	14.83	–	–	155.3	20.26	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	162.0	4.54

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

Remarks:           DMSO (200 µL) was applied as solvent of the test item and DMSO (100 µL) was applied as solvent of the positive control substances 9AA, NPD and 2AA. The ultrapure water (100 µL) was applied as solvent of the positive control substances MMS and SAZ. The mutation rate of the test item and the untreated control refers to the DMSO sample (200 µL); the mutation rate of the 9AA, NPD and 2AA refers to the DMSO sample (100 µL). The mutation rate of MMS and SAZ refers to ultrapure water (100 µL).

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

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

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

                               TA1537;E. coli:Escherichia coliWP2uvrA

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

Conclusions:

The test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. The study included preliminary solubility tests, preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content (86.65 % with rounding 87 %) 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; 16 and 5 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 precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye. When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentration range of 5000-500 µg/plate in the absence and at 5000 and 1600 µg/plate in presence of S9 following the plate incorporation and pre-incubation procedures. An inhibitory effect of the test item was observed in the initial mutation test in the S. typhimurium TA1537 strain, in the confirmatory mutation test in the S. typhimurium TA98, TA100 and TA1537 strains in the absence and also presence of exogenous metabolic activation (slight inhibition was noticed in TA1535, in absence of S9). The inhibitory effect was indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges) and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 500 µg/plate (noticed in S. typhimurium TA98 and TA1537) was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of solvent control (dimethyl sulfoxide (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants 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 item has no mutagenic activity on the applied bacterium tester strains 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:

key study

Study period:

06 March 2018 - 23 March 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:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

Sub-line (K1)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- Suitability of cells: suitable cell line for this type of assay

MEDIA USED
- Type and identity of media including CO2 concentration: 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.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

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:

Concentrations:
- Main test with metabolic activation: 39.1, 78.2, 156.3, 234.4 and 312.5 µg/mL
- Main test without metabolic activation: 39.1, 78.2, 156.3, 312.5, 625 and 937.5 µg/mL
Justification for top dose: based on preliminary cytotoxicity test

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: This solvent was chosen based on the results of the preliminary solubility test.

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
- Cell density at seeding: 5 x 10E6 cells/dish

DURATION
- Preincubation period: 24 hours
- Exposure duration: 5 hours
- Expression time: 8 days
- Selection time: not specified

SELECTION AGENT: selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG)

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 /dish

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

According to Guideline

Evaluation criteria:

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,
• 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 item 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)

Remarks:

Sub-line (K1)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES:
The concentrations for the main mutation assay were selected on the basis of preliminary cytotoxicity investigations (without and with metabolic activation using S9-mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control.

Please see attached document.

Conclusions:

The test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.

Executive summary:

The test item suspended in DMSO, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD guideline 476. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver: 39.1, 78.2, 156.3, 234.4 and 312.5 µg/mL (without S9-mix) and 39.1, 78.2, 156.3, 312.5, 625 and 937.5 µg/mL (with S9-mix).Phenotypic expression was evaluated up to 8 days following exposure.

There was no precipitation of the test item 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 and presence of metabolic activation clear cytotoxicity (survival between 17-19 %) of the test item was observed at the highest concentration applied (312.5 µg/mL in the absence and 937.5 µg/mL in the presence of S9 mix). In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit).The mutation frequency found in the solvent controls was in the 95% confidence interval of the historical control data. The concurrent positive controls ethyl methanesulfonate (1.0 µL/mL) and 7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.The test itemtested up to cytotoxic concentrations with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and 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 performed with Chinese hamster ovary cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 September 2017 - 23 March 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:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammallian chromosome aberration test

Target gene:

Chromatid and chromosome type aberrations (gaps, deletions and exchanges), in metaphase cells.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79: Chinese hamster lung male
Lot. No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)

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). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
This cell line was purchased from ECACC (European Collection of Cells Cultures). 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. The V79 cells for this study were grown in 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 g/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.

Cytokinesis block (if used):

Cell cultures were treated with colchicine (0.2 µg/mL) 2.5 hours prior to harvesting.

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation of substances was achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were 33.7 and 33.8 mg/mL.

Test concentrations with justification for top dose:

A homogeneous suspension of Blue Sema was obtained in DMSO up to a concentration of 100 mg/mL.

Based on the results of the cytotoxicity assay the following concentrations were selected for the chromosome aberration assay:

Experiment A with 3/20 h treatment/sampling time
without S9 mix: 31.3, 62.5, 125 and 250 g/mL test item
with S9 mix: 31.3, 62.5, 125 and 250 *g/mL test item

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 7.8, 15.7, 31.3 and 62.5 g/mL test item

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 7.8, 15.7, 31.3 and 62.5 g/mL test item

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 31.3, 62.5, 125 and 250 g/mL test item

Vehicle / solvent:

Dimethyl sulfoxide DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

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.
Mammalian Microsomal Fraction S9 Mix

An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential when they are metabolised by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were 33.7 and 33.8 mg/mL.

Rat Liver S9 Fraction

The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2, D-35394 Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189, BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier. The Certificate of Analysis of rat liver S9 mix is stored in the laboratory.

S9 Mix (with Rat Liver S9)

The complete S9 Mix was freshly prepared containing components with the following ratios:
S9 fraction 3 mL
HEPES* 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP** 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL
*= N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**= β-Nicotinamide Adenine Dinucleotide Phosphate
Before adding to the culture medium the S9 mix was kept in an ice bath.

Rationale for test conditions:

Acceptability of the Assay

The chromosome aberration assay is considered acceptable because it meets the following criteria:
– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data,
– concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control,
– cell proliferation in the solvent control is adequate,
– adequate number of cells and concentrations are analyzable,
– all requested experimental conditions were tested unless one resulted in a positive result
– the criteria for the selection of top concentration are fulfilled.

Evaluation criteria:

Evaluation of Result

Treatment of results
– 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility

A homogeneous suspension of Blue Sema was obtained in DMSO up to a concentration of 100 mg/mL. There was no precipitation in the medium at any concentration tested.

All concentrations were run in duplicates (incl. negative and positive controls) and 300 (150 per culture) well-spread metaphases were assessed.

Chromosome Aberration Assay

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.

In both experiments, clear cytotoxicity of about 50% was observed after test item treatment in the absence and presence of metabolic activation.

No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation .

In experiment A in the absence of metabolic activation, two values (5 aberrant cells excluding gaps/150 cells) were slightly above the 95% control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells). 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. No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of Blue Sema.

The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µL/mL) and Cyclophosphamide (5 µg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data . Thus, the study is considered valid.

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

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	–	2100000	2100000	1968750	-	-	-
	-	B	–	1900000	1900000
	-	C	–	2050000	1850000
	-	D	–	1950000	1900000
Solvent control (DMSO)	-	A	–	7650000	7700000	7800000	5831250	100,00	0,00
	-	B	–	8000000	7850000
Blue Sema	31.3	A	–	7750000	7450000	7600000	5631250	96,57	3,43
	62.5	A	–	6550000	6650000	6600000	4631250	79,42	20,58
	125	A	–	5100000	5400000	5250000	3281250	56,27	43,73
	250	A	–	4600000	4500000	4550000	2581250	44,27	55,73
	500	A	–	4150000	4100000	4125000	2156250	36,98	63,02
	EMS 1 µL/mL	A	–	4950000	4750000	4850000	2881250	49,41	50,59
Solvent control (DMSO)	-	A	+	7500000	7700000	7562500	5593750	100,00	0,00
	-	B	+	7600000	7450000
Blue Sema	31.3	A	+	7300000	7300000	7300000	5331250	95,31	4,69
	62.5	A	+	6000000	5900000	5950000	3981250	71,17	28,83
	125	A	+	5600000	5450000	5525000	3556250	63,58	36,42
	250	A	+	4450000	4500000	4475000	2506250	44,80	55,20
	500	A	+	3850000	4050000	3950000	1981250	35,42	64,58
	Cycl. 5µg/mL	A	+	4500000	4600000	4550000	2581250	46,15	53,85

RICC=Relative Increase in Cell Counts

Cytotoxicity= 100-RICC

EMS: Ethyl methanesulfonate (EMS)

Cycl: Cyclophosphamide monohydrate

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

 

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	–	2100000	2100000	1968750	-	-	-
	-	B	–	1900000	1900000
	-	C	–	2050000	1850000
	-	D	–	1950000	1900000
Solvent control (DMSO)	-	A	–	7450000	7750000	7562500	5593750	100,00	0,00
	-	B	–	7550000	7500000
Blue Sema	31.3	A	–	5950000	5750000	5850000	3881250	69,39	30,61
	62.5	A	–	4500000	4400000	4450000	2481250	44,36	55,64
	125	A	–	3600000	3800000	3700000	1731250	30,95	69,05
	250	A	–	3100000	2800000	2950000	981250	17,54	82,46
	500	A	–	2050000	2250000	2150000	181250	3,24	96,76
	EMS 1 µL/mL	A	–	4600000	4750000	4675000	2706250	48,38	51,62

RICC=Relative Increase in Cell Counts

Cytotoxicity= 100-RICC

EMS: Ethyl methanesulfonate (EMS)

 

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

 

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	–	2100000	2100000	1968750	-	-	-
	-	B	–	1900000	1900000
	-	C	–	2050000	1850000
	-	D	–	1950000	1900000
Solvent control (DMSO)	-	A	–	9100000	9400000	9250000	7281250	100,00	0,00
	-	B	–	9150000	9350000
Blue Sema	31.3	A	–	7250000	7100000	7175000	5206250	71,50	28,50
	62.5	A	–	5300000	5250000	5275000	3306250	45,41	54,59
	125	A	–	4100000	4250000	4175000	2206250	30,30	69,70
	250	A	–	3500000	3450000	3475000	1506250	20,69	79,31
	500	A	–	3100000	2950000	3025000	1056250	14,51	85,49
	EMS 1 µL/mL	A	–	5500000	5650000	5575000	3606250	49,53	50,47
Solvent control (DMSO)	-	A	+	9600000	9650000	9550000	7581250	100,00	0,00
	-	B	+	9550000	9400000
Blue Sema	31.3	A	+	9350000	9200000	9275000	7306250	96,37	3,63
	62.5	A	+	7250000	7350000	7300000	5331250	70,32	29,68
	125	A	+	6800000	6600000	6700000	4731250	62,41	37,59
	250	A	+	5400000	5500000	5450000	3481250	45,92	54,08
	500	A	+	4800000	5050000	4925000	2956250	38,99	61,01
	Cycl. 5µg/mL	A	+	5600000	5700000	5650000	3681250	48,56	51,44

RICC=Relative Increase in Cell Counts

Cytotoxicity= 100-RICC

EMS: Ethyl methanesulfonate (EMS)

MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) 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	4
Blue Sema
31.3 µg/mL	-	3 h	20 h	7	4
62.5 µg/mL	-	3 h	20 h	9	5
125 µg/mL	-	3 h	20 h	8	4
250 µg/mL	-	3 h	20 h	9	5
Pos. Control (Ethyl methanesulphonate)	-	3 h	20 h	39**	34**
Negative (Solvent) control	+	3 h	20 h	8	4
Blue Sema
31.3 µg/mL	+	3 h	20 h	8	4
62.5 µg/mL	+	3 h	20 h	9	4
125 µg/mL	+	3 h	20 h	8	3
250 µg/mL	+	3 h	20 h	8	3
Pos. Control (Cyclophosphamide)	+	3 h	20 h	45**	38**

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

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

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

MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) 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	6	4
	Blue Sema
	7.8 µg/mL	-	20 h	20 h	8	4
	15.7 µg/mL	-	20 h	20 h	8	4
	31.3 µg/mL	-	20 h	20 h	6	3
	62.5 µg/mL	-	20 h	20 h	7	3
	Pos. Control (Ethyl methanesulphonate)	-	20 h	20 h	49**	43**
	Negative (Solvent) control	-	20 h	28 h	6	3
	Blue Sema
	7.8 µg/mL	-	20 h	28 h	9	4
	15.7 µg/mL	-	20 h	20 h	7	4
	31.3 µg/mL	-	20 h	28 h	8	3
	62.5 µg/mL	-	20 h	28 h	10	4
	Pos. Control (Ethyl methanesulphonate)	-	20 h	28 h	44**	36**

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

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

 

MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) 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	7	3
Blue Sema
31.3 µg/mL	+	3 h	28 h	7	3
62.5 µg/mL	+	3 h	28 h	9	4
125 µg/mL	+	3 h	28 h	8	4
250 µg/mL	+	3 h	28 h	9	4
Pos. Control (Cyclophosphamide)	+	3 h	28 h	46**	34**

Cyclophosphamide: 5.0g/mL

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

 

APPENDIX IV

 

NUMBER OF POLYPLOID 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
Blue Sema
31.3 µg/mL	-	3/20 h	0.0	0.0
62.5 µg/mL	-	3/20 h	0.0	0.0
125 µg/mL	-	3/20 h	0.0	0.0
250 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
Blue Sema
31.3 µg/mL	+	3/20 h	0.0	0.0
62.5 µg/mL	+	3/20 h	0.0	0.0
125 µg/mL	+	3/20 h	0.0	0.0
250 µg/mL	+	3/20 h	0.0	0.0
Pos. Control (Cyclophosphamide)	+	3/20 h	0.0	0.0

Ethyl methanesulphonate: 1.0mL/mL

Cyclophosphamide: 5.0g/mL

 

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

TABLE 11

 

NUMBER OF POLYPLOID 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
Blue Sema
7.8 µg/mL	-	20/20 h	0.0	0.0
15.7 µg/mL	-	20/20 h	0.0	0.0
31.3 µg/mL	-	20/20 h	0.0	0.0
62.5 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
Blue Sema
7.8 µg/mL	-	20/28 h	0.0	0.0
15.6 µg/mL	-	20/28 h	0.0	0.0
31.3 µg/mL	-	20/28 h	0.0	0.0
62.5 µg/mL	-	20/28 h	0.0	0.0
Pos. Control	-	20/28 h	0.0	0.0

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

 

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

 

NUMBER OF POLYPLOID 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
Blue Sema
31.3 µg/mL	+	3/28 h	0.0	0.0
62.5 µg/mL	+	3/28 h	0.0	0.0
125 µg/mL	+	3/28 h	0.0	0.0
250 µg/mL	+	3/28 h	0.0	0.0
Pos. Control	+	3/28 h	0.0	0.0

Cyclophosphamide: 5.0g/mL

 

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

 

 

pH AND OSMOLALITY DATA

Pre-test on Toxicity (Concentration selection)

 

Concentration (µg/mL)		pH	Osmolality (mmol/kg)
3-hour treatment /without S9 Mix/20-hour sampling times
Negative (solvent) Control		7.96	409
Blue Sema	31.3	8.01	409
	62.5	8.01	416
	125	8.03	411
	250	8.03	415
	500	8.04	415
	EMS	8.04	356
20-hour treatment /without S9 Mix/20 and 28-hour sampling times
Negative (solvent) Control		7.96	409
Blue Sema	31.3	8.01	409
	62.5	8.01	416
	125	8.03	411
	250	8.03	415
	500	8.04	415
	EMS	8.04	356
3-hour treatment with S9 Mix / 20 and 28-hour sampling time
Negative (solvent) Control		7.96	406
Blue Sema	31.3	7.97	412
	62.5	7.94	414
	125	7.93	416
	250	7.97	413
	500	7.94	412
	Cycl.	7.83	339

EMS:Ethyl methanesulphonate(1L/mL, 0.4L/mL )

Cycl.:Cyclophosphamide (5.0g/mL)

 

 

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		7.97	373
Blue Sema	31.3	8.06	376
	62.5	8.07	374
	125	8.09	374
	250	8.09	377
	EMS	8.05	353
3-hour treatment with S9 Mix / 20-hour sampling time
Negative (solvent) Control		7.99	372
Blue Sema	31.3	8.00	374
	62.5	8.02	375
	125	8.02	373
	250	8.04	376
	Cycl.	8.00	357

EMS:Ethyl methanesulphonate(1L/mL)

Cycl.:Cyclophosphamide (5.0g/mL)

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		7.92	352
Blue Sema	7.8	7.96	351
	15.7	8.12	350
	31.3	8.10	352
	62.5	8.14	354
	EMS	8.09	349
3-hour treatment with S9 Mix / 28-hour sampling time
Negative (solvent) Control		7.94	374
Blue Sema	31.3	8.02	378
	62.5	8.03	377
	125	8.07	376
	250	8.12	378
	Cycl.	8.03	353

EMS:Ethyl methanesulphonate(0.4L/mL )

Cycl.:Cyclophosphamide (5.0g/mL)

Conclusions:

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.

Executive summary:

The test item suspended in DMSO was tested in a chromosome aberration assay in V79 cells in two independent experiments according to OECD guideline 473. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix): 31.3, 62.5, 125 and 250g/mL test item for Experiment A with 3/20 h treatment/sampling time and Experiment B with 3/28 h treatment/sampling time and 7.8, 15.7, 31.3and 62.5g/mL test item for Experiment B with 20/20 h and 20/28 h treatment/sampling time. Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). 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 absence of metabolic activation, two 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 L/mL) and cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. The study included preliminary solubility tests, preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content (86.65 % with rounding 87 %) 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; 16 and 5 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 precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye. When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentration range of 5000-500 µg/plate in the absence and at 5000 and 1600 µg/plate in presence of S9 following the plate incorporation and pre-incubation procedures. An inhibitory effect of the test item was observed in the initial mutation test in the S. typhimurium TA1537 strain, in the confirmatory mutation test in the S. typhimurium TA98, TA100 and TA1537 strains in the absence and also presence of exogenous metabolic activation (slight inhibition was noticed in TA1535, in absence of S9). The inhibitory effect was indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges) and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 500 µg/plate (noticed in S. typhimurium TA98 and TA1537) was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of solvent control (dimethyl sulfoxide (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants 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 item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

OECD 476:

The test item suspended in DMSO, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD guideline 476. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver: 39.1, 78.2, 156.3, 234.4 and 312.5 µg/mL (without S9-mix) and 39.1, 78.2, 156.3, 312.5, 625 and 937.5 µg/mL (with S9-mix). Phenotypic expression was evaluated up to 8 days following exposure. There was no precipitation of the test item 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 and presence of metabolic activation clear cytotoxicity (survival between 17-19 %) of the test item was observed at the highest concentration applied (312.5 µg/mL in the absence and 937.5 µg/mL in the presence of S9 mix). In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit).The mutation frequency found in the solvent controls was in the 95% confidence interval of the historical control data. The concurrent positive controlsethyl methanesulfonate (1.0 µL/mL) and7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid. The test item tested up to cytotoxic concentrations with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and 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 performed with Chinese hamster ovary cells.

OECD 473:

The test item suspended in DMSO was tested in a chromosome aberration assay in V79 cells in two independent experiments according to OECD guideline 473. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix): 31.3, 62.5, 125 and 250g/mL test item for Experiment A with 3/20 h treatment/sampling time and Experiment B with 3/28 h treatment/sampling time and 7.8, 15.7, 31.3and 62.5g/mL test item for Experiment B with 20/20 h and 20/28 h treatment/sampling time. Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). 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 absence of metabolic activation, two 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 L/mL) and cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the 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.

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.