Reaction product of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. and Benzenesulfonic acid, 4-methyl- and sodium hydroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

May 25, 1995-November 23, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study done according to OECD guidelines. However, this study does not adequately address the results obtained at mildly cytotoxic concentrations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, C10-13 alkyl derivatives, sodium salt

IUPAC Name:

Benzenesulfonic acid, C10-13 alkyl derivatives, sodium salt

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

All concentrations in micrograms/ml

Test 1 with S9: 0.32, 0.63, 1.25, 2.5, 5, 10, 20, 39, 78
Test 1 without S9: 1.25, 2.5, 5, 10, 20, 39, 58,78, 156

Test 2 with S9: 2.5, 5, 10, 20, 26, 33, 39
Test 2 without S9: 20, 39, 58, 78, 130, 156

An additional test was done with S9 at the following dose levels:
2.5, 5, 7.5, 10, 15, 20, 25, and 30 ug/ml

Vehicle / solvent:

None

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methyl methanesulphonate, cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 6 hrs with S9, 22 hrs without S9
- Expression time (cells in growth medium): 16-40 hrs with S9, 40 hrs without S9
- Selection time (if incubation with a selection agent): 2 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 24-48 hrs


SELECTION AGENT (mutation assays): Colcemid

NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 100 metaphases


DETERMINATION OF CYTOTOXICITY
- Method: number of cells per culture


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A dosage was considered toxic if cell count was less then 60% of cell cultures. A test substance was considered clastogenic if a single dose caused the percentage of aberrant cells to be consistently greater than the 99% confidence limits of negative controls and there was also an increase at another dose level.

Statistics:

95% and 99% confidence limits

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 15 microgram/ml with S9, >=58 microgram/ml without S9

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence of S9, only one culture (Test 2, 24 hr harvest, 20 ug/ml) showed a suspicious result. This single result was considered sporadic, as other cultures at this concentration, or at higher concentrations did not show a positive response. In Test 1, in the absence of S9, cytoxicity was seen at 78 micrograms/ml and above. In Test 2, in the absence of S9, cytoxicity was seen at concentrations of 58 micrograms/ml and above.

In Test 1, in the presence of S9, no positive results were seen at concentrations of up to 20 micrograms/ml. Metaphases could not be analyzed due to severe cytotoxicity at the 39 and 78 microgram/ml concentrations. In Test 2, in the presence of S9, one of the cultures at the 5 microgram/ml concentration gave a suspicious result, and both cultures at the 10 microgram/ml concentrations gave positive responses. Mild cytotoxity was also seen at the 10 microgram/ml concentration. At concentrations at and above 20 micrograms/ml, metaphases could not be analyzed due to severe cytotoxicity. No positive results were seen in the Test 2, 48 hr harvest cultures grown in the presence of S9, though moderate cytotoxicity was seen in one of the 20 microgram/ml cultures, and severe cytotoxicity was seen in all cultures above this concentration.

A third test was done in the presence of S9, which showed positive results at the 15 micrograms/ml concentration. However, this concentration was also moderately cytotoxic with only 26% of cells survival. However, due to the low survival of cells, these results are not definitive for determining clastogenicity. Higher concentrations were completely cytotoxic. An additional assessment was then performed at 10 micrograms/ml in the presence of S9, with negative results.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Abbreviations used in tables:

T ¿ Toxicity evident from morphological changes

TT- Toxicity evident from reduced cell count (<60% of vehicle)

TTT- Too toxic for metaphase assessment

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham¿s F10 medium	0.01	1	0	Nil
Ham¿s F10 medium	0.02	1	1	Nil
0.32	-	-	-	Nil
0.32	-	-	-	Nil
0.63	-	-	-	Nil
0.63	-	-	-	Nil
1.25	-	-	-	Nil
1.25	-	-	-	Nil
2.5	0.01	1	0	Nil
2.5	0.00	0	0	Nil
5	0.00	0	0	Nil
5	0.05	5	0	Nil
10	0.01	1	0	Nil
10	0.01	1	0	Nil
20	0.00	0	0	Nil
20	0.00	0	0	Nil
39	-	-	-	TTT
39	-	-	-	TTT
78	-	-	-	TTT
78	-	-	-	TTT
Cyclophosphamide (20 micrograms/ml)	0.14	8	4	-
Cyclophosphamide (30 micrograms/ml)	0.06	4	4	-
Cyclophosphamide (40 micrograms/ml)	0.33	20	19	-

Test 1 ¿ Without S9 Mix, 24 hr Harvest

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham¿s F10 medium	0.00	0	0	Nil
Ham¿s F10 medium	0.00	0	0	Nil
1.25	-	-	-	Nil
1.25	-	-	-	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
10	-	-	-	Nil
10	-	-	-	Nil
20	-	-	-	Nil
20	-	-	-	Nil
39	0.01	1	0	Nil
39	0.00	0	0	Nil
58	0.01	1	0	Nil
58	0.00	0	0	Nil
78	0.00	0	0	T
78	0.00	0	0	T
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (10 micrograms/ml)	0.03	3	1	-
Cyclophosphamide (20 micrograms/ml)	0.16	14	10	-

Test 2 ¿ With S9 Mix, 24 hr Harvest

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham¿s F-10 medium	0.01	1	0	Nil
Ham¿s F-10 medium	0.02	2	1	Nil
2.5	0.07	2	1	Nil
2.5	0.04	3	1	Nil
5	0.04	3	2	Nil
5	0.06	6	4	Nil
10	0.12	8	6	T
10	0.19	13	5	T
20	-	-	-	TTT
20	-	-	-	TTT
26	-	-	-	TTT
26	-	-	-	TTT
33	-	-	-	TTT
33	-	-	-	TTT
39	-	-	-	TTT
39	-	-	-	TTT
Cyclophosphamide (40 micrograms/ml)	0.38	20	17	-
Cyclophosphamide (50 micrograms/ml)	0.31	18	11	-

Test 2 ¿ With S9 Mix, 48 hr Harvest

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham¿s F-10 medium	0.00	0	0	Nil
Ham¿s F-10 medium	0.00	0	0	Nil
2.5	0.01	1	0	Nil
2.5	0.01	1	1	Nil
5	0.00	0	0	Nil
5	0.02	2	2	Nil
10	0.03	2	1	Nil
10	0.02	2	1	TT
20	-	-	-	TTT
20	-	-	-	TTT
26	-	-	-	TTT
26	-	-	-	TTT
33	-	-	-	TTT
33	-	-	-	TTT
39	-	-	-	TTT
39	-	-	-	TTT
Cyclophosphamide (40 micrograms/ml)	0.03	3	2	-
Cyclophosphamide (50 micrograms/ml)	0.10	8	7	-

Test 2 ¿ Without S9 Mix, 24 hr Harvest

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham¿s F-10 medium	0.02	2	2	Nil
Ham¿s F-10 medium	0.03	3	0	Nil
20	0.02	2	0	Nil
20	0.05	5	3	Nil
39	0.02	2	1	Nil
39	0.04	4	0	Nil
58	0.01	1	1	Nil
58	0.06	6	1	Nil
78	-	-	-	TTT
78	-	-	-	TTT
104	-	-	-	TTT
104	-	-	-	TTT
130	-	-	-	TTT
130	-	-	-	TTT
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (10 micrograms/ml)	0.30	21	14	-
Methyl methane-sulphonate (20 micrograms/ml)	0.71	33	28	-

Test 2 ¿ Without S9 Mix, 48 hr Harvest

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Ham¿s F-10 medium	0.01	1	1	Nil
Ham¿s F-10 medium	0.00	0	0	Nil
20	0.00	0	0	Nil
20	0.00	0	0	Nil
39	0.01	1	1	Nil
39	0.00	0	0	Nil
58	0.00	0	0	T
58	0.01	1	0	T
78	-	-	-	TTT
78	-	-	-	TTT
104	-	-	-	TTT
104	-	-	-	TTT
130	-	-	-	TTT
130	-	-	-	TTT
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (20 micrograms/ml)	0.21	11	8	-
Methyl methane- sulphonate (40 micrograms/ml)	3.20	60	60	-

Test 3 ¿ With S9 Mix, 24 hr Harvest

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytoxicity
Ham¿s F-10 medium	0.04	4	0	Nil
Ham¿s F-10 medium	0.04	4	0	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
7.5	-	-	-	Nil
7.5	-	-	-	Nil
10	-	-	-	Nil
10	-	-	-	Nil
15	0.20	12	8	TT
15	0.18	12	6	TT
20	-	-	-	TTT
20	-	-	-	TTT
25	-	-	-	TTT
25	-	-	-	TTT
30	-	-	-	TTT
30	-	-	-	TTT
Cyclophosphamide (30 micrograms/ml)	0.24	14	12	-
Cyclophosphamide (40 micrograms/ml)	0.32	17	11	-

Test 3 - see tables below

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
positive with metabolic activation at cytotoxic concentrations or above

The test substance is not clastogenic in the absence of metabolic activation. The test substance is also not clastogenic in the presence of metabolic activation at non-cytotoxic concentrations. At cytotoxic concentrations, the test substance is weakly clastogenic.

Executive summary:

This study examined the potential of the test substance Marlon A 350 to cause chromosomal aberrations in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 0.32 to 78 ug/ml with S9, and 1.25 to 156 ug/ml without S9. Methyl methanesuflphonate and cyclophosphamide were used as positive controls. No biologically significant results were seen in treated cultures in the absence of metabolic activation. Positive responses were seen at cytotoxic concentrations in the presence of S9. Concentrations below the level of cytotoxicty with S9 did not show positive results. The test substance is not clastogenic in the absence of metabolic activation, or with metabolic activation below cytotoxic concentrations. These results indicate that LAS is weakly clastogenic at cytotoxic concentrations but negative at concentrations below cytotoxic concentrations