Reaction mass of dipotassium 3,3'-sulphonylbis(benzenesulphonate) and potassium 3-(phenylsulphonyl)benzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In recent GLP guideline studies conducted to examine the genetic toxicity (mutagenicity and chromosome abberations) of the test substance in vitro in bacterial cells and mammalian cells (Chinese hamster V79 cells), both with and without S9 metabolic activation, the substance does exhibit positive genotoxic effects in the mammalian assays. In a mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells, with and without S9 metabolic activation, the substance showed no signs of mutagenicity without metabolic activation up to and including doses that produced biologically relevant growth inhibition but was shown to be mutagenic with metabolic activation only at the highest dose tested. In a mammalian cell chromosome abberation test in Chinese Hamster V79 cells the substance did not induce structural chromosomal aberrations in the short-term assay but did induce structural chromosomal aberrations in the long-term assay.

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:

26 July 1990 - 17 September 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Includes most, but not all, currently recommended bacterial strains

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Includes most, but not all, currently recommended bacterial strains

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Includes most, but not all, currently recommended bacterial strains

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Includes most, but not all, currently recommended bacterial strains

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test Substance Name: DPS
Product Number: 016513
Appearance: White powder
Storage: Room temp
Chemical name: Diphenylsulfone-3-sulfonic acid, Potassium-salt
Content:
- 86.5% DPS
- 5.4% By-product I
- 1.6% By-product III
- 0.8% Diphenylsulfone
- 4.2% H2O

Note: The substance described above is believed to be the same or similar to the reaction mass being registered but was not described as such in 1991.

Target gene:

Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Test concentrations used were 8, 40, 200, 1000 and 5000 ug/Plate

Vehicle / solvent:

The solvent employed was deionized water and, for the positive controls, DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See Remarks

Remarks:

Positive Controls (without S9): sodium azide (TA1535); nitrofurantoin (TA100); 4-nitro-1,2-phenylene diamine (TA1537, TA98); Postive Controls (with S9) 2-aminoantrachene

Details on test system and experimental conditions:

For the mutant count, four plates were used, both with and without 59 mix, for each strain and dose. The same number of plates, filled with thee solvent minus the test substance, comprised the negative control. Each positive control also contained four plate per strain. The doses for the first trial were routinely determined on the basis of a standard protocol: 5000 ug or 5 ul per plate were used as the highest dose, if not limited by solubility. At least four additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. In case of a positive response, however, or if at least three doses could be evaluated for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first <0 experiment.

Evaluation criteria:

The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

An assay which did not comply with at least on of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 1537

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

The Salmonella/microsome test, employing doses up to 5000 ug per plate, showed the substance not to produce bacteriotoxic or mutagenic effects. Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility) revealed no biologically relevant variations from the respective negative controls. No inhibition of growth was noted even at the limit dose. Positive controls, at comparatively low doses, resulted in the expected increases of the mutant counts to well over double those of the negative controls, and thus demonstrated the system's high sensitivity. Despite this sensitivity, no indications of mutagenic effects could be found at assessable doses up to 5000 ug per plate in any of the Salmonella typhimurium strains used.

Conclusions:

The test item is not matagenic in the bacterial reverse mutation assay.

Executive summary:

In a GLP guideline bacterial reverse mutation assay using the plate incorporation method conducted according to OPPTS 870.5100, OECD 471, and EC method B.13/14, the substance showed no indications of mutagenic effects at doses up to 5000 uq per plate in any of the Salmonella typhimurium strains used.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 February 2018 - 03 April 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

Name: KSS-FR
Chemical Name: Potassium 3-(phenylsulphonyl)benzenesulphonate
Batch No.: KSS-1711-02
Composition: 70.8% KSS (CAS No.: 63316-43-8)
20.9% DKSS (CAS No.: 63316-33-6)
8.3% other
pH: 7.11
Physical State: powder
Color: white
Expiry Date: November 2019
Storage Conditions: at room temperature
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety

Target gene:

Hprt and xprt genes

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

exogenous metabolic

Test concentrations with justification for top dose:

Without Metabolic activation: 2000, 2500, 2750 and 3250 µg/mL
With metabolic activation: 2250, 2750, 3000 and 3500 µg/mL

Vehicle / solvent:

No

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Not Applicable

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins BioPharma Product Testing Munich GmbH. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (via PCR), stable spontaneous mutant frequency as well as stability of the modal chromosome number. Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37°C incubation temperature. For purifying the cell population of pre-existing HPRT- mutants cells were exposed to HAT medium containing 10 µM hypoxanthine, 3.2 µM aminopterin, 5 µM thymidine and 10 µM glycine for several cell doublings (2-3 days) with a subsequent recovery period in medium supplemented with 10 µM hypoxanthine and 5 µM thymidine.

Approx. 2 - 6 million cells per treatment group were seeded in complete culture medium in a 175 cm2 culture flask. Approx. 24 h after seeding, the cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation. After 4 h the cultures were checked for precipitation and the treatment medium containing the test item (MEM without FBS) was removed. The cells were washed twice with PBS, trypsinised and counted with a cell counter. During the following expression period most of the cells were subcultured in complete culture medium (MEM supplemented with 10% FBS) in a sufficient number of cells (at least 2 x 106 cells per treatment group). In addition, for determination of the relative survival (RS) two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium for each treatment group. After incubation for an appropriate time (6 - 7 days) colonies were fixed with methanol, stained with Giemsa and counted. Cytotoxicity (relative survival) was calculated based on the cloning efficiency of cells plated immediately after treatment adjusted by any loss of cells during treatment.

Rationale for test conditions:

V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14 h doubling time of the Eurofins BioPharma Product Testing Munich GmbH stock cultures) and their high cloning efficiency of untreated cells, usually more than 50%. These facts are necessary for the appropriate performance of the study.

Evaluation criteria:

A test chemical is considered to be 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

A test chemical 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, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Highest dose only.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the experiment without and with metabolic activation all validity criteria were met. The mutant values of the negative controls fall within the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls are > 50%. The positive controls, DMBA (1.0 µg/mL) and EMS (300 µg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.

Main Experiment – Toxicity, without metabolic activation

Dose Group	Concen-tration [µg/mL]	Number of cells at the		Number of colonies per flask			CE[%]	Adjusted CE [%]	Relative Survival (RS) [%]
		beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	9588000	191	173	182	91	87	100
NC2		10000000	10268000	163	185	174	87	89
1	500	10000000	10132000	133	141	137	69	69	79
2	1000	10000000	11628000	104	109	107	53	62	70
3	1500	10000000	10336000	116	129	123	61	63	72
4	2000	10000000	11560000	94	130	112	56	65	73
5	2500	10000000	10676000	203	152	178	89	95	107
6	2750	10000000	10812000	37	45	41	21	22	25
7	3000	20000000	18564000	10	11	11	5	5	6
8	3250	20000000	11050000	17	34	26	13	7	8
EMS	300	10000000	13005000	145	120	133	66	86	98

NC:negative control; CE:cloning efficiency; EMS:Ethylmethanesulfonate

Main Experiment – Mutagenicity, without metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration [µg/mL]	Number of colonies per flask			CE[%]	Number of colonies per flask							CE[%]	Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	178	160	169	85	1	4	5	5	2	3.4	1.6	0.0009	10.1
NC2		185	157	171	86	6	7	8	6	7	6.8	0.7	0.0017	19.9
4	2000	158	168	163	82	7	6	6	7	4	6.0	1.1	0.0015	18.4
5	2500	161	163	162	81	6	4	5	4	2	4.2	1.3	0.0011	13.0
6	2750	162	160	161	81	2	8	4	11	8	6.6	3.2	0.0017	20.5
8	3250	150	157	154	77	10	5	10	7	6	7.6	2.1	0.0019	24.8
EMS	300	161	175	168	84	80	74	91	78	93	83.2	7.5	0.0208	247.6

NC:negative control; CE:cloning efficiency; EMS:Ethylmethanesulfonate

Main Experiment – Toxicity, with metabolic activation

Dose Group	Concen-tration [µg/mL]	Number of cells at the		Number of colonies per flask			CE[%]	Adjusted CE [%]	Relative Survival (RS) [%]
		beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	13464000	162	156	159	80	107	100
NC2		10000000	13396000	137	134	136	68	91
1	500	10000000	13617000	130	122	126	63	86	87
2	750	10000000	13991000	117	136	127	63	88	89
3	1000	10000000	11951000	126	119	123	61	73	74
4	2250	10000000	9197000	122	121	122	61	56	56
5	2750	10000000	9214000	103	93	98	49	45	46
6	3000	10000000	9163000	74	78	76	38	35	35
7	3500	20000000	16014000	57	38	48	24	19	19
8	4000	20000000	15640000	18	19	19	9	7	7
9	4250	20000000	14654000	6	4	5	3	2	2
10	4500	20000000	10914000	3	0	2	1	0	0
DMBA	1.0	10000000	13073000	171	171	171	86	112	113

NC:negative control; CE:cloning efficiency; DMBA:7,12-dimethylbenz(a)anthracene

Main Experiment – Mutagenicity, with metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration [µg/mL]	Number of colonies per flask			CE[%]	Number of colonies per flask							CE[%]	Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	162	159	161	80	12	13	10	9	16	12.0	2.4	0.0030	37.4
NC2		141	147	144	72	8	9	16	7	9	9.8	3.2	0.0025	34.0
4	2250	153	146	150	75	8	10	7	12	8	9.0	1.8	0.0023	30.1
5	2750	161	163	162	81	15	12	12	10	5	10.8	3.3	0.0027	33.3
6	3000	157	159	158	79	11	11	6	10	8	9.2	1.9	0.0023	29.1
7	3500	149	139	144	72	12	18	12	14	12	13.6	2.3	0.0034	47.2
DMBA	1.0	144	143	144	72	101	115	100	118	99	106.6	8.2	0.0267	371.4

NC:negative control; CE:cloning efficiency; DMBA:7,12-dimethylbenz(a)anthracene

Conclusions:

The test item was considered to be mutagenic only at the highest dose tested in the in vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells.

Executive summary:

In this GLP guideline study conducted according to OECD 476, EU Method B.17 and OPPTS 870.5300 to assess the test item's potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster, with and without S9 metabolic activation, the substance showed no signs of mutagenicity without metabolic activation up to and including doses that produced biologically relevant growth inhibition but was shown to be mutagenic with metabolic activation only at the highest dose tested. Positive controls showed the expected biologically relevant effects in mutation frequency, thus supporting the validity of the study. Given these results, the test item should be considered mutagenic in the in vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells and this result should be weighed accordingly with other in vitro and any in vivo data to determine the substance's mutagenicity potential.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2018 - 30 May 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: KSS-FR
Chemical Name: Potassium 3-(phenylsulphonyl)benzenesulphonate
Batch No.: KSS-1711-02
Composition: 70.8% KSS (CAS No.: 63316-43-8)
20.9% DKSS (CAS No.: 63316-33-6)
8.3% other
pH: 7.11
Physical State: powder
Color: white
Expiry Date: November 2019
Storage Conditions: at room temperature

Target gene:

Entire Chromosome

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Experiment I (without metabolic activation): 1000, 2000 and 2500 µg/mL
Experiment I (with metabolic activation): 2500, 3000 and 3500 µg/mL
Experiment II (without metabolic activation): 500, 1000 and 1500 µg/mL

Vehicle / solvent:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Not Applicable

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

V79 cells in vitro are widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are chosen because of their relatively small number of chromosomes (diploid number, 2n = 22), their high proliferation rate (doubling time of the Eurofins Munich V79 in stock cultures: 12 - 14 h) and a high plating efficiency of untreated cells (normally more than 50%). These facts are necessary for the appropriate performance of the study.

The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections was carried out before freezing.

For the experiment thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10E5 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made 3-4 days after seeding.

Complete Culture Medium: MEM medium supplemented with:
- 10% (v/v) Fetal bovine serum (FBS)
- 100 U/100 µg/mL penicillin/streptomycin solution
- 2 mM L-glutamine
- 2.5 µg/mL amphotericin
- 25 mM HEPES
- Also used for the long-term treatment and the post incubation.

Treatment Medium (short-term exposure): Complete culture medium without FBS.

The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et al.

Three or four days old stock cultures (in exponential growth) more than 50% confluent were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment. Cells subsequently were trypsinised with a solution of 0.05% trypsin in Ca-Mg-free PBS at 37 °C for about 5 min. By adding complete culture medium the detachment was stopped and a single cell suspension was prepared. About 1 x 104 cells/mL were seeded into cell culture flasks with complete culture medium.

Treatment parameters/conditions were as follows:

Exp. I (without S9 mix):
- Treatment Period: 4 hours
- Recovery Time: 17 hours
- Preparation interval: 21 hours
Exp. II (without S9 mix):
- Treatment Period: 21 hours
- Recovery Time: TBD
- Preparation interval: 21 hours
Exp. I (with S9 mix):
- Treatment Period: 4 hours
- Recovery Time: 17 hours
- Preparation interval: 21 hours

Rationale for test conditions:

A pre-experiment was conducted under identical conditions as described for the main experiment. The following concentrations were tested without and with S9 mix: 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL. The results of this pre-experiment were used to select the concentrations for the main experiment.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the 95% control limits of the historical negative control data.

When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined

a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the 95% control limits of the historical negative control data.

The test chemical is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline. For the trend test, Statistical significance at the 5% level (p < 0.05) was evaluated by the x² test. The p value was used as a limit in judging for significance levels

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

@1000 ug/mL and higher

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

@1000 ug/mL and higher

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity: In experiment I without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 3500 µg/mL and higher (58% at 3500 µg/mL and 6% at 4000 µg/mL. With metabolic activation, no biologically relevant decrease of the RICC was observed. In experiment II without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 1000 µg/mL and higher (53% at 1000 µg/mL, 48% at 1500 µg/mL, 25% at 2000 µg/mL, 11% at 2500 µg/mL and 0% at 3000 µg/mL.

Clastogenicity: In experiment I without metabolic activation, the aberration rate of the negative control (1.7%) and two test item concentrations were within the historical control data of the testing facility (-0.28 to 3.70% aberrant cells exclusive gaps). A slight induction of aberrant cells was noted at a concentrations of 2000 µg/mL (4.0%). However, this increase was considered as not biologically relevant due to the lack of a dose response relationship. With metabolic activation, the aberration rates of the negative control (2.7%) and all dose groups treated with the test item were within the historical control data of the testing facility (-0.23 to 3.95% aberrant cells exclusive gaps. In experiment II without metabolic activation, the aberration rate of the negative control (1.3%) and the lowest evaluated concentration of 500 µg/mL (0.7%) were within the historical control data of the testing facility (-0.20 to 2.71% aberrant cells exclusive gaps). A dose-dependent increase of aberrant cells was noted in the two next higher concentrations of 1000 µg/mL (5.7%) and 1500 µg/mL mM (16.4%). The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the experiment I without and with metabolic activation. In experiment II, the concentrations 1000 µg/mL and 1500 µg/mL which were above the historic control range, were also statistically significantly increased. The X² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in experiment I without and with metabolic activation. However, a statistically significant dose-dependent increase was noted in experiment II without metabolic activation. EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

Polyploid Cells: No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Experiment I (short-term) - Summary of Cytotoxicity Data

Dose Group	Concentration [µg/mL]	Cell Count				Precipitate (+/-)
		Culture			RICC
		1	2	Mean	[%]
without metabolic activation
C	0	160.20	174.84	167.52	100	-
1	1000	119.20	158.24	138.72	82	-
2	2000	146.53	134.81	140.67	83	-
3	2500	121.15	140.67	130.91	77	+
4	3000	127.98	130.91	129.45	76	+
5	3500	94.70	107.48	101.09	58	+
6	4000	23.44	16.61	20.02	6	+
EMS	600	143.60	162.15	152.87	91	-
with metabolic activation
C	0	142.62	204.12	173.37	100	-
1	2000	235.36	175.81	205.59	120	-
2	2500	155.31	172.89	164.10	94	-
3	3000	155.31	170.93	163.12	94	-
4	3500	173.86	155.31	164.59	95	+
5	4000	186.55	87.87	137.21	78	+
6	4500	137.74	78.11	107.92	60	+
7	5000	72.25	29.30	50.77	25	+
CPA	0.83	44.91	162.15	103.53	57	-

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the control groups. The cell count was determined by a cell counter per culture for each test group.

C: Negative Control (Culture Medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

CPA: Positive Control (with metabolic activation: Cyclophosphamide)

Experiment I – Summary of Aberration Rates

Dose Group	Concentration (µg/mL)	Treatment Time (hour)	Fixation Interval	Mean % Abeerrant Cells		Precipitation (+/-)
				incl. Gaps	excl. Gaps
without metabolic activation
C	0	4	21	3.0	1.7	-
1	1000	4	21	4.7	2.7	-
2	2000	4	21	5.3	4.0	-
3	2500	4	21	3.3	2.0	+
EMS	600	4	21	9.3	8.0	-
with metabolic activation
C	0	4	21	5.3	2.7	-
2	2500	4	21	5.0	3.3	-
3	3000	4	21	4.3	1.3	-
4	3500	4	21	4.3	2.7	+
CPA	0.83	4	21	14.2	12.9	-

300 cells evaluated for each concentration, except for the positive control CPA (225 cells) due to a clearly positive increase in chromosomal aberrations.

C: Negative Control (Culture Medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

CPA: Positive Control (with metabolic activation: Cyclophosphamide)

Experiment II - Summary of Cytotoxicity Data

Dose Group	Concentration [µg/mL]	Cell Count				Precipitate (+/-)
		Culture			RICC
		1	2	Mean	[%]
without metabolic activation
C	0	215.00	217.00	216.00	100	-
S	0	215.00	217.00	216.00	100	-
1	500	236.00	226.00	231.00	107	-
2	1000	107.00	131.00	119.00	53	-
3	1500	98.00	118.00	108.00	48	-
4	2000	59.00	63.00	61.00	25	-
5	2500	28.00	39.00	33.50	11	-
6	3000	1.90	4.70	3.30	0	-
EMS	400	189.00	180.00	184.50	85	-

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the control groups. The cell count was determined by a cell counter per culture for each test group.

C: Negative Control (Culture Medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

Experiment II - Summary of Aberration Rates

Dose Group	Concentration (µg/mL)	Treatment Time (hour)	Fixation Interval	Mean % Abeerrant Cells		Precipitation (+/-)
				incl. Gaps	excl. Gaps
without metabolic activation
C	0	21	21	3.3	1.3	-
1	500	21	21	1.0	0.7	-
2	1000	21	21	8.3	5.7	-
3	1500	21	21	18.4	16.4	-
EMS	400	21	21	18.0	16.0	-

300 cells evaluated for each concentration, except for the positive controls (EMS: 150 cells) and the concentration 1500 µg/mL (250 cells) due to a clearly positive increase in chromosomal aberrations.

C: Negative Control (Culture Medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

Conclusions:

The test substance induced structural chromosomal aberrations in the V79 Chinese hamster cell line in the long-term experiment.

Executive summary:

In this GLP guideline study, conducted according to OECD 472, EC method B.10, and US EPA Method OPPTS 870.5375, to investigate in vitro the potential for the test substance to induce structural chromosome abberations in chinese hamster V79 cells the substance did not induce structural chromosomal aberrations in the short-term assay (Experiment I) but did induce structural chromosomal aberrations in the long-term assay (Experiment II).

In experiment I without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 3500 µg/mL and higher (58% at 3500 µg/mL and 6% at 4000 µg/mL. With metabolic activation, no biologically relevant decrease of the RICC was observed. The aberration rate of the negative control (1.7%) and two test item concentrations were within the historical control data of the testing facility (-0.28 to 3.70% aberrant cells exclusive gaps). A slight induction of aberrant cells was noted at a concentrations of 2000 µg/mL (4.0%). However, this increase was considered as not biologically relevant due to the lack of a dose response relationship. With metabolic activation, the aberration rates of the negative control (2.7%) and all dose groups treated with the test item were within the historical control data of the testing facility (-0.23 to 3.95% aberrant cells exclusive gaps. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the experiment I without and with metabolic activation.

In experiment II without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 1000 µg/mL and higher (53% at 1000 µg/mL, 48% at 1500 µg/mL, 25% at 2000 µg/mL, 11% at 2500 µg/mL and 0% at 3000 µg/mL. The aberration rate of the negative control (1.3%) and the lowest evaluated concentration of 500 µg/mL (0.7%) were within the historical control data of the testing facility (-0.20 to 2.71% aberrant cells exclusive gaps). A dose-dependent increase of aberrant cells was noted in the two next higher concentrations of 1000 µg/mL (5.7%) and 1500 µg/mL mM (16.4%). These concentrations were statistically significantly increased and were found to be statistically significant dose-dependent increases.

No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

Given these results, the test substance did induce structural chromosomal aberrations in the V79 Chinese hamster cell line in the long-term experiment and the need for in vivo testing should be considered.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The relevance of these in vitro findings to humans is unknown.

Justification for classification or non-classification

Despite the positive findings in vitro, the substance does not meet the GHS criterial for classification as a Germ Cell Mutagen on the basis that there is no positive evidence in vivo in either germ cells or somatic cells and there is no known chemical structure activity relationship to known germ cell mutagens.