Sulfonic acids, C6-8-alkane, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria: Negative ± S9 (OECD TG 471)

In vitro chromosome aberration assay: Negative ± S9 (OECD TG 473)

In vitro mammalian cell gene mutation test: Negative ± S9 (OECD TG 476)

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:

10 October 2012-10 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and Tryptophan

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 rat liver induced with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Range-finding test (TA 98, TA100): 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate
Main tests: 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Substance is soluble in water

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:

METHOD OF APPLICATION: in agar (plate incorporation) Experiment 1; preincubation Experiment 2

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per duplicate experiment

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of background lawn of bacteria

Evaluation criteria:

Criteria for a Positive Response:
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:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

According to the OECD guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

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

Statistics:

Not applicable.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: None
- Water solubility: Soluble
- Precipitation: None
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: Range-finder experiment performed to set the dose levels for the two main experiments.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all control data within the historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity varied slightly between bacterial strains; more toxic to strains TA1535 and TA1537.

Summary Table of the Initial Mutation Test and Complementary Initial Mutation Test

 

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	25.7	20.7	99.0	120.3	37.0	22.0	7.0	6.3	28.0	37.0
	MF	0.99	0.98	0.99	0.99	1.19	1.43	1.17	1.19	0.79	0.81
DMSO control	Mean	27.3	18.7	--	112.7	--	21.0	8.3	8.7	--	44.3
	MF	1.05	0.89	--	0.93	--	1.37	1.39	1.63	--	0.97
Distilled water control	Mean	26.0	21.0	100.3	121.7	31.0	15.3	6.0	5.3	35.7	45.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	20.7	30.0	99.0	127.3	21.0	14.3	4.3	8.0	37.0	38.3
	MF	0.79	1.43	0.99	1.05	0.68	0.93	0.72	1.50	1.04	0.84
1581	Mean	16.3	29.7	101.3	122.7	17.0	10.3	5.7	6.3	31.7	36.3
	MF	0.63	1.41	1.01	1.01	0.55	0.67	0.94	1.19	0.89	0.80
500	Mean	18.3	22.3	98.7	114.0	20.7	16.3	4.3	7.0	37.3	35.7
	MF	0.71	1.06	0.98	0.94	0.67	1.07	0.72	1.31	1.05	0.78
158.1	Mean	17.0	24.7	101.7	112.3	18.3	14.0	7.7	6.7	33.7	40.7
	MF	0.65	1.17	1.01	0.92	0.59	0.91	1.28	1.25	0.94	0.89
50	Mean	17.0	23.3	107.3	117.3	17.0	12.3	5.7	9.7	30.0	42.3
	MF	0.65	1.11	1.07	0.96	0.55	0.80	0.94	1.81	0.84	0.93
15.81	Mean	16.0	28.7	106.7	119.7	15.3	17.0	6.0	9.7	36.3	42.3
	MF	0.62	1.37	1.06	0.98	0.49	1.11	1.00	1.81	1.02	0.93
5	Mean	22.7	30.3	109.3	131.7	16.7	19.0	6.0	9.7	36.7	37.7
	MF	0.87	1.44	1.09	1.08	0.54	1.24	1.00	1.81	1.03	0.82
NPD (4µg)	Mean	321.0	--	--	--	--	--	--	--	--	--
	MF	11.74	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2276.0	--	2111.3	--	201.0	--	213.0	--	--
	MF	--	121.93	--	18.74	--	9.57	--	24.58	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	247.7
	MF	--	--	--	--	--	--	--	--	--	5.59
SAZ (2µg)	Mean	--	--	1210.7	--	1173.3	--	--	--	--	--
	MF	--	--	12.57	--	37.85	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	466.7	--	--	--
	MF	--	--	--	--	--	--	56.00	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1203.3	--
	MF	--	--	--	--	--	--	--	--	33.74	--

 

 

Conclusions:

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 October 2012 to 31 January 2012

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not relevant

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: Chromosome medium 1 with phytohaemagglutinin (Gibco)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable

Metabolic activation:

with and without

Metabolic activation system:

BNF-PG induced rat liver S9

Test concentrations with justification for top dose:

0, 125, 250, 500, 1000, 1500, 2000, 2500 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Bioterger was soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 20 and 0 hours
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per duplicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Other:

OTHER:

Evaluation criteria:

The test item is judged to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:
o the number of chromosomal aberrations is significantly (at p  0.05) increased compared with the solvent control
o the increase observed is concentration-dependent
o both duplicate cultures provide similar results
o the increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity may cause artefacts in the form of aberrations in in vitro chromosomal aberration tests
o a reproducible increase in the number of cells with chromosomal aberrations.

Statistics:

The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p < 0.05) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, Statistical evaluation of mutagenicity test data, 1989).

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: Not relevant
- Water solubility: Soluble
- Precipitation: None
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: Range finder experiment performed

COMPARISON WITH HISTORICAL CONTROL DATA: Control values within the historical range

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Concentration of Bio-Terge PAS 7S-DRY [µg/mL medium]	pH value	osmolality [mOsmol/kg]
Negative control	7.51	280.5
125	7.51	276.0
250	7.51	285.5
500	7.52	295.0
1000	7.46	293.5
1500	7.50	293.5
2000	7.45	295.0
2500	7.64	299.0

The results of the first and second experiments are provided in the attached tables and no statistically significant increases in the frequency of cells with aberrations were observed, either in the presence or absence of metabolic activation.

Conclusions:

Under the present test conditions, C6-8 alkane sulfonate, tested up to a cytotoxic concentration of 2500 µg /mL medium, in the absence and in the presence of metabolic activation, employing two exposure times (without S9) and one exposure time (with S9), revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.
In the same test, Mitomycin C and cyclophosphamide induced significant increases in the frequency of damaged cells, which confirmed the validity of this assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 September - 14 October 2019

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 using the Hprt and xprt genes)

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

Specific details on test material used for the study:

Batch/Lot number: 0008282300
Description: Clear liquid
Purity: 40.9 % in water
Expiry date: 13 July 2021
Storage conditions: Controlled room temperature (15-25 °C, ≤70% relative humidity)

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Sub-line (K1) of Chinese hamster ovary cell line CHO; ATCC No. CCL-61; Lot No. 58244452; American Type Culture Collection (Manassas,Virginia)
- Karyotype: Chromosome Frequency Distribution 50 Cells: 2n = 22. Stemline number is hypodiploid.

For cell lines:
- Absence of Mycoplasma contamination: Tested negative
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: the cells were diluted in F12-10 medium and incubated in a humidified atmosphere (at 37°C (± 0.5°C), 5± 0.3% CO2 in air).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : prepared from male Wistar rat liver by the Microbiological Laboratory of Charles River Laboratories Hungary Kft.
- method of preparation of S9 mix : Constituent concentrations (in mL/mL) HEPES - 0.2; KCl - 0.1; MgCl2 - 0.1; NADP - 0.1; D-glucose-6-phosphate - 0.1; F12-10 - 0.1; S9 fraction - 0.3.
- concentration or volume of S9 mix and S9 in the final culture medium: For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to 9 mL of cell culture medium to give a total of 10 mL (the same ratio was applied in those cases when higher treatment volume was used). The final concentration of the liver homogenate in the test system was 3%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The sterility of the preparation was confirmed. The protein concentration was determined by colorimetric test by chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Charles River Laboratories Hungary Kft. The protein concentration of the S9 fraction used was determined to be 30.45 g/L. The biological activity in a Salmonella assay of each batch of S9 was characterized using 2-Aminoanthracene and another mutagen, Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batches of S9 used in this study functioned appropriately during the activity checking.

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assays were selected based on the result of a short preliminary toxicity test. The highest test concentration in the preliminary test was 2000 μg/mL (the maximal recommended concentration).

Assay 1:
5 hours (+S9; 100 μL/mL): 2000, 666.67, 222.22, 74.07, 24.69, 8.23 μg/mL
5 hours (-S9): 2000, 666.67, 222.22, 74.07, 24.69, 8.23 μg/mL
Assay 2:
5 hours (+S9; 100 μL/mL): 2000, 666.67, 222.22, 74.07, 24.69, 8.23 μg/mL
24 hours (-S9): 2000, 666.67, 222.22, 74.07, 24.69, 8.23 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water used for test item; DMSO used for positive control

- Justification for choice of solvent/vehicle: Based on the results of a short solubility test, distilled water as vehicle was suitable for the test.

Untreated negative controls:

yes

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): at least 2x10^6 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: approximately 24 hour
- Exposure duration/duration of treatment: 5 or 24 hours
After the 5-hour incubation period the cultures were washed thoroughly with F12-10 medium then covered with the appropriate amount of fresh F12-10 medium and incubated for approx 19 hours. After the 19-hour incubation period, cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x105 cells/mL. Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.
After the 24-hour incubation period in a humidified atmosphere (at 37°C (± 0.5°C), 5± 0.3% CO2 in air), cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x105 cells/mL. Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days
- Selection agent: 6-thioguanine; concentration: 10 μg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 4x105 cells/mL. After the growing or selection period, the culture medium was removed and colonies were fixed for 5 minutes with methanol. After fixation, colonies were stained using 10% Giemsa solution (diluted with distilled water) for 30 minutes, dried and manually counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x105 cells/mL, then further diluted to 40 cells/mL using F12-10 medium. Five mL of cell suspension (200 cells/dish) per each culture were plated in F12-10 medium in 3 parallel dishes (diameter was approx. 60 mm) for a viability test. The dishes were incubated in a humidified atmosphere (at 37°C (± 0.5°C), 5± 0.3% CO2 in air) for 5 days for colony growing.

OTHER
Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.
The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x106 cells: 5 plates at 4x105 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 106 clonable cells.

Rationale for test conditions:

Based on dose recommended in TG when no precipitate or limiting toxicity is observed.

Evaluation criteria:

The test item was considered to be mutagenic in this assay if the following criteria are met (in line with the OECD No. 476 TG):
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
5. The historical control range is considered when deciding if the result is positive.
Results which only partially met the criteria were dealt with on a case-by-case basis (the historical control data of untreated control samples were taken into consideration if necessary).
According to the OECD No. 476 guideline, the biological relevance of the results was considered first, i.e. statistical significance was not the only determinant for a positive response.

Statistics:

The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The homogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a linear trend in mutation frequency with treatment dose using Microsoft Excel software (R-squared values were calculated for the log concentration versus the mutation frequency).
In the statistical analysis, negative trends were not considered significant.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There was no significant change in pH when the test item was dosed into media at 8.23 to 2000 μg/mL.
- Data on osmolality: There was no large increase in osmolality during the study.
- Possibility of evaporation from medium: no
- Water solubility: Soluble in water
- Precipitation and time of the determination: No precipitation observed

RANGE-FINDING/SCREENING STUDIES (if applicable): Treatment concentrations for the mutation assays were selected based on the results of a short preliminary experiment. A 5-hour treatment, in the presence or absence of S9-mix, and a 24-hour treatment, in the absence of S9-mix, was performed with a range of test item concentrations to determine potential toxicity immediately after the treatments. The highest test concentration in the preliminary test was 2000 μg/mL. Tabulated results of the preliminary experiment are given in Appendix 3 (attached). No cytotoxicity was detected in the preliminary experiment. The concentrations selected for the main experiments were as suggested in the OECD No. 476 TG. Lower test concentrations were separated by factor of three. Six concentrations were selected for the main experiments with or without metabolic activation.

STUDY RESULTS
Assay 1
In Assay 1, a 5-hour treatment with metabolic activation (in the presence of S9-mix) and a 5-hour treatment without metabolic activation (in the absence of S9-mix) were performed.
For the 5-hour treatment in the presence and absence of S9-mix, the following concentrations were examined: 2000, 666.67, 222.22, 74.07, 24.69 and 8.23 μg/mL.

In Assay 1 no insolubility was detected in the final treatment medium at the end of the treatment in the experiment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
In the presence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence of a dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.

In the absence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence of a dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.

Data of Assay 1 are presented for survival (Table 1 of Appendix 4 and Appendix 7), viability (Table 3 of Appendix 5 and Appendix 9) and mutagenicity (Table 5 of Appendix 6 and Appendix 11). Observations after treatment are summarized in Appendix 13.

Assay 2
In Assay 2, 5-hour treatment with metabolic activation (in the presence of S9-mix) and 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed.
For the 5-hour treatment in the presence of S9-mix, and for 24-hour treatment in the absence of S9-mix, the following concentrations were examined: 2000, 666.67, 222.22, 74.07, 24.69 and 8.23 μg/mL.

In Assay 2 no insolubility was detected in the final treatment medium at the end of the treatment in the experiments with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
In Assay 2, in the presence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence of a dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.

In Assay 2, in the absence of S9-mix (24-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. Statistically significant increases in mutant frequencies (at p<0.05 level or p<0.01 level) were observed in the concentration range of 2000-8.23 μg/mL with the exception of 24.69 μg/mL (see Table 6). However, the observed values were within the historical control data. Furthermore, the observed mutant frequency values (6.4 x 10^-6 at 2000 μg/mL; 10.2 x 10^-6 at 666.67 μg/mL, 7.2 x 10^-6 at 222.22 μg/mL, 8.9 x 10^-6 at 74.07 μg/mL and 7.4 x 10^-6 at 8.23 μg/mL) were within the expected range of the negative control samples according to the OECD No. 476 TG (expected range: 5-20 x 10^-6). There was no evidence for a dose-response relationship (a trend analysis showed no effect of treatment). This experiment is considered to be negative.

Data from Assay 2 are presented for survival (Table 2 of Appendix 4 and Appendix 8), viability (Table 4 of Appendix 5 and Appendix 10) and mutagenicity (Table 6 of Appendix 6 and Appendix 12). Observations after treatment are summarized in Appendix 13.
All the observed mutation frequency values were consistent with the general historical control range and in line with the OECD guideline*. Taken together with the lack of a correlation with dose level, this demonstrates that there were no biologically significant differences between treated samples and negative (vehicle) controls.
*Note: The spontaneous mutant frequency is generally between 5 and 20 x10^-6 based on the guideline.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data) :Appendix 15 attached
- Positive historical control data: Mutation frequency (Number of 6-TG resistant mutants per 106 clonable cells): DMBA 5-hour, S9+; mean 905.2; SD 562.7; range 141.2 - 2119.4; n = 27; EMS 5-hour, S9-; mean 445.6; SD 118.6; range 239.6 - 636.6; n = 13; EMS 24-hour, S9-; mean 1176.6; SD 610.9; range 363.1 - 2449.8; n = 14
- Negative (solvent/vehicle) historical control data: Mutation frequency (Number of 6-TG resistant mutants per 106 clonable cells): Distilled water 5-hour, S9+; mean 11.5; SD 3.8; range 6.1-15.8; n=6; Distilled water 5-hour, S9-; mean 9.1; SD 3.4; range 5.2-11.6; n=3; Distilled water 24-hour, S9-; mean 15.5; SD 5.6; range 9.2 - 20.1; n=3; DMSO 5-hour, S9+; mean 21.8; SD 15.9; range 5.4-57.3; n=29; DMSO 5-hour, S9-; mean 18.9; SD 11.6; range 6.5 - 47.4; n=13; DMSO 24-hour, S9-; mean 18.4; SD 14.4; range 6.8-48.5; n=14
utant Frequencies per survivor (x 10^-6): 13.92 ± 7.04 (-S9); 20.62 ± 13.64 (+S9)

Executive summary:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to evaluate the potential of the test item to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix).

Distilled water was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:

Assay 1

5-hour treatment in the presence and in the absence of S9-mix:

2000, 666.67, 222.22, 74.07, 24.69 and 8.23 μg/mL

Assay 2

5-hour treatment in the presence of S9-mix:

2000, 666.67, 222.22, 74.07, 24.69 and 8.23 μg/mL

24-hour treatment in the absence of S9-mix:

2000, 666.67, 222.22, 74.07, 24.69 and 8.23 μg/mL

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.

In Assay 1 and Assay 2 no insolubility was detected in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.

In Assay 1, in the presence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence of dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.

In Assay 1, in the absence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence of dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.

In Assay 2 with metabolic activation no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence of dose response to the treatment (a trend analysis showed no effect of treatment).

The experiments with metabolic activation are considered to be negative.

In Assay 2, in the absence of S9-mix (24-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. Statistically significant increase of mutant frequencies (at p<0.05 level or p<0.01 level) was observed at concentration range of 2000-8.23 μg/mL with the exception of 24.69 μg/mL (see Table 5), although the observed values were within the historical control data range. Furthermore, the observed mutant frequency values were within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6). Moreover, there was no evidence for a dose-response relationship and trend analysis demonstrated no effect of treatment. This experiment is considered to be negative.

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in compatible with the historical data in all assays. Six evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested in accordance with the recommendation of the OECD TG 476 in each test). The study was considered to be valid.

In conclusion, no mutagenic effect of the test item was observed either in the presence or absence of a metabolic activation system under the conditions of this OECD TG 476 HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Mutation Assay

Alkane C6 -C8 (even numbered), 1-sulphonic acid, sodium salt (C6-8 alkane sulfonate) was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Complementary Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Solubility Test and available information, the test item was dissolved in Distilled water. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 or 10 µg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81 or 5 μg/plate. In the Initial Mutation Tests and Confirmatory Mutation Tests none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect. In the Confirmatory Mutation Tests inhibitory or cytotoxic effect of the test item was observed in Salmonella typhimurium TA98 and TA100 bacterial strains at 5000μg/plate concentration without metabolic activation and in Salmonella typhimurium TA1535 and TA1537 bacterial strains at 5000 and 1581μg/plate concentration without metabolic activation. The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests. The tests were considered to be valid. 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, C6-8 alkane sulfonate had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.

Chromosome Aberration Test in Mammalian Cells

Test samples of C6-8 alkane sulfonate were assayed in an in vitro cytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after starting of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate. C6-8 alkane sulfonate was completely dissolved in aqua ad iniectabilia. A correction factor of 1.02 was used as the purity of C6-8 alkane sulfonate was 98% only. The vehicle served as the negative control.

A preliminary cytotoxicity study was conducted to establish the top concentration for the main cytogenetic test. Concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg C6-8 alkane sulfonate/mL medium were employed in an experiment without and with metabolic activation.In this preliminary experiment cytotoxicity was noted at 2500 and 5000 µg C6-8 alkane sulfonate /mL in the experiment with and without metabolic activation (4-h or 24-h exposure). Hence, the highest concentration employed in the main study was 2500 µg C6-8 alkane sulfonate /mL medium in the experiments without and with metabolic activation.

In the main study cytotoxicity was noted in the experiments without and with metabolic activation at the top concentration of 2500 µg C6-8 alkane sulfonate /mL medium. Mitomycin C and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively.

Tests without metabolic activation (4- and 24-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with C6-8 alkane sulfonate at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 µg C6-8 alkane sulfonate /mL medium (4-h or 24-h exposure) in the absence of metabolic activation ranged from 1.0% to 3.5%. These results were within the range of the historical control data (0 - 4%).

The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, which was in line with the historical control range. Therefore, the test is considered to be valid.

Test with metabolic activation (4-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with C6-8 alkane sulfonate at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 µg C6-8 alkane sulfonate /mL medium in the presence of metabolic activation in the first and second experiment ranged from 0.5% to 4.0%. These results were within the range of the historical control data (0 - 4%).

The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, which was in line with the historical control range. Therefore, the test is considered to be valid. No test item-related polyploidy or endoreduplication were noted in the experiments without or with metabolic activation.

In the same test, Mitomycin C and cyclophosphamide induced significant increases in the frequency of damaged cells, which confirmed the validity of this assay.

Conclusion

Under the present test conditions, C6-8 alkane sulfonate, when tested up to a cytotoxic concentration of 2500 µg C6-8 alkane sulfonate /mL medium, in the absence and in the presence of metabolic activation, employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.

In Vitro Mammalian Cell Gene Mutation Test

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to evaluate the potential of the test item to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix).

Distilled water was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:

Assay 1

5-hour treatment in the presence and in the absence of S9-mix:

2000, 666.67, 222.22, 74.07, 24.69 and 8.23 μg/mL

Assay 2

5-hour treatment in the presence of S9-mix:

2000, 666.67, 222.22, 74.07, 24.69 and 8.23 μg/mL

24-hour treatment in the absence of S9-mix:

2000, 666.67, 222.22, 74.07, 24.69 and 8.23 μg/mL

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.

In Assay 1 and Assay 2 no insolubility was detected in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.

In Assay 1, in the presence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence of dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.

In Assay 1, in the absence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence of dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.

In Assay 2 with metabolic activation no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence of dose response to the treatment (a trend analysis showed no effect of treatment).

The experiments with metabolic activation are considered to be negative.

In Assay 2, in the absence of S9-mix (24-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all six concentrations. Statistically significant increase of mutant frequencies (at p<0.05 level or p<0.01 level) was observed at concentration range of 2000-8.23 μg/mL with the exception of 24.69 μg/mL (see Table 5), although the observed values were within the historical control data range. Furthermore, the observed mutant frequency values were within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6). Moreover, there was no evidence for a dose-response relationship and trend analysis demonstrated no effect of treatment. This experiment is considered to be negative.

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were incompatible with the historical data in all assays. Six evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested in accordance with the recommendation of the OECD TG 476 in each test). The study was considered to be valid.

In conclusion, no mutagenic effect of the test item was observed either in the presence or absence of a metabolic activation system under the conditions of this OECD TG 476 HPRT assay.

Justification for classification or non-classification

Based on the available data, no additional classification is proposed. All three in vitro genotoxicity assays were negative.