Sulfuric acid, mono-C16-20-alkyl esters, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay: III. 72 Coded chemicals

Author:

McGregor, D.B. et al.

Year:

1988

Bibliographic source:

Environmental and Molecular Mutagenesis 12:85-154

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

lack of details on test substance

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium dodecyl sulphate

EC Number:

205-788-1

EC Name:

Sodium dodecyl sulphate

Cas Number:

151-21-3

Molecular formula:

C12H26O4S.Na

IUPAC Name:

sodium dodecyl sulfate

Method

Target gene:

Thymidine kinase locus (tk)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fisher’s medium supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/mL 0.05% pluronic F68, antbiotics and 10% heat-inactivated donor horse serum (v/v)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Fischer 344 rats, intraperitoneally induced with Arochlor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Experiments 1-5: -S9: 3.125, 6.25, 10, 12.5, 20, 25, 30, 40, 50, 55, 60,65, 70, 80 and 100 µg/mL
Experiments 6-8: +S9: 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 µg/mL

Vehicle / solvent:

- Vehicle used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: methylmethanesulfonate (MMS), 15 µg/mL; +S9: 3-methylcholanthrene (3-MCA), 2.5 µg/mL

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h with and without S9 mix
- Expression time: 2 days
- Selection time: 11-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-16 days

SELECTION AGENT: 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: four cultures for vehicle control; two cultures for positive controls and each test substance concentration

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

Four response categories for evaluation of results were defined (see below).

Response Categories for Experiments:
Positive response (+): The dose-related trend and the response at one of the three highest acceptable doses were statistically significant.
Negative response (-) Two categories were used. In both there was
a) no dose-related trend,
b) no statistically significant response at any dose,
c) an acceptable positive control response.
Nontoxic, negative response ( = )
There was an RTG among the acceptable doses of >30% (approximately), higher toxicities being unattainable due to intrinsic properties of either the compound or the system.
Toxic, negative response (-)
There was either an RTG of <30% (approximately) at the maximum acceptable dose, or the lethal concentration was no greater than 1.5 x a lower concentration at which the RTG was >30%.
Inconclusive (i)
There was
a) no dose-related trend and a statistically significant dose was any other than one of the highest three doses,
b) a response which would have been negative, but the lowest RTG acceptable doses was >35%,
c) a response which would have been negative, but there were no acceptable positive controls.
Questionable (?)
There was either
a) no dose-related trend, but a statistically significant response occurred at one of the highest three doses, or
b) a statistically significant dose-related trend, but none of the acceptable doses was statistically significant on its own.

Primary judgments were made at the level of individual experiments, but judgment on the mutagenic potential of a chemical was made on a basis of consensus of all valid experimental results (see "Any other information on materials and methods inlc. tables").

Statistics:

The statistical analysis was based upon the mathematical model proposed for this system and consisted of a dose-trend test and a variance analysis of pair-wise comparisons of each dose against the vehicle control. Significant differences from concurrent vehicle control values are indicated at the 5% level.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9: 70, 80 and 90 µg/mL; +S9: 95 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

RESULTS OF EXPERIMENTS 1-8

Eight acceptable experiments were conducted, five in the absence of S9 mix.

In the first of these, statistically significant increases in mutant fraction were observed at three dose levels: 6.25, 25, and 50 µg/mL; 100 µg/mL was a lethal concentration in cells (see Table 1). Over the nonlethal range, there were generally elevated mutant fractors, the highest being 1.9-fold the control level at 25 µg/mL. Although these increases in mutant fraction were significant, the lack of an obvious dose-related response with a relatively soluble chemical over a dose range which was not toxic encouraged speculation that the increases were not due to treatment with the test material.

 

Table 1. Experiment 1 - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency	Relative Total Growth	Mutants per 1E+06 surviving cells	Mutation factor	Average Mutation factor
DMSO (NC)	62	95	56	30	43
	68	98	90	44
	65	95	66	34
	80	112	153	64
3.125	77	106	84	36	61
	59	97	151	85
6.25	71	119	176	83	78*
	73	107	160	74
12.5	90	145	133	49	65
	67	133	160	80
25	84	90	247	99	83*
	65	88	130	67
50	86	82	192	75	69*
	76	98	145	64
100	lethal	lethal	n.a.	n.a.	n.a.
			n.a.	n.a.
MMS (15 µg/mL) PC	27	21	135	167	232*
	25	23	219	298

MMS = methylmethanesulfonate; NC = negative control; PC = positive control; *p < 0.05; n.a. = not applicable

 

In the second experiment without S9 mix, there was a clearly significant response at 60 µg/mL, but at no other concentration. The RTG was about 22%

Table 2. Experiment II - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency	Relative Total Growth	Mutants per 1E+06 surviving cells	Mutation factor	Average Mutation factor
DMSO (NC)	80	100	115	48	48
	74	108	119	53
	86	106	115	45
	61	87	83	45
10	75	112	107	47	58
	66	94	137	69
20	67	89	119	59	52
	53	76	70	44
30	71	70	98	46	60
	64	86	144	75
40	77	77	126	54	n.a.
50	94	61	191	68	74
	68	56	163	80
60	81	27	365	150	203*
	77	16	595	256
70	lethal	lethal	n.a.	n.a.	n.a.
			n.a.	n.a.
MMS (15 µg/mL) PC	34	30	683	666	664*
	29	27	573	662

MMS = methylmethanesulfonate; NC = negative control; PC = positive control; *p < 0.05; n.a. = not applicable

Experiment 3 gave a statistically significant response (1.7-fold increase) at 60 µg/mL, but not at the next higher concentration of 65 µg/mL. The mutant fraction at 70 µg/ml was only 44/106 survivors, so this single culture result supported the view that the statistically significant result at the lower dose level was a chance event. Thus, this experiment was judged to be questionable.

 

Table 3. Experiment 3 - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency	Relative Total Growth	Mutants per 1E+06 surviving cells	Mutation factor	Average Mutation factor
DMSO (NC)	76	103	84	37	34
	71	104	73	34
	82	97	98	40
	74	96	58	26
50	60	71	65	36	36
	75	75	82	36
55	68	44	68	33	38
	53	71	69	44
60	72	63	107	50	56*
	72	74	134	62
65	64	71	87	45	42
	79	68	93	39
70	80	83	107	44	n.a.
MMS (15 µg/mL) PC	36	26	127	119	158*
	26	23	156	197

MMS = methylmethanesulfonate; NC = negative control; PC = positive control; *p < 0.05; n.a. = not applicable

 

However, the succeeding experiments 4 and 5 without S9 mix were unambiguously negative; therefore the test substance was considered to be non-mutagenic in the absence of S9 mix.

Two experiments (6 and 7) were performed in the presence of S9 mix, showing unambiguously negative results. The last experiment with S9 mix was inconclusive because the cloning efficiency at 80 µg/mL was about 86% and there was no indication of a mutagenic response. However, based on the two experiments with S9 mix showing clearly negative results, the test substance was considered to be not mutagenic in the presence of S9 mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Executive summary:

No enhanced mutation rate in the S9 treated or untreated cells was observed in this mouse lymphoma assay. Therefore, the test substance was not considered to be mutagenic.