Alcohols, C12-14, ethoxylated, sulfates, 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

Study period:

10 Oct. 1994 - 25 Nov. 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C12-14, ethoxylated, sulfates, sodium salts

EC Number:

500-234-8

EC Name:

Alcohols, C12-14, ethoxylated, sulfates, sodium salts

Cas Number:

68891-38-3

Molecular formula:

not applicable, UVCB

IUPAC Name:

Alcohols, C12-14(even numbered), ethoxylated < 2.5 EO, sulfates, sodium salts

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK+/- mouse lymphoma cells; obtained form American Type Culture Collection, Rockville, MD
- Suitability of cells: recommended in TG 476
- Absence of Mycoplasma contamination: yes
- generation time, plating efficiency and mutation rates (spontaneous and induced) were checked

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 minimal medium supplemented with 10% horse serum heat-inactivated at 56°C for 20 min before use (complete medium). All incubations at 37°C in a 5% CO2 atmosphere (100% humidity).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of five young male Sprague-Dawley rats treated with phenobarbitone and betanaphthoflavone.
- quality controls of S9: The efficacy was checked in an Ames test and produced acceptable responses with 2-aminoanthracene and benzo(a)pyrene in S. typhimurium TA100; protein content (28.4 ± 0.37 and 24.2 ± 4.19 mg/mL) and aminopyrine demethylase activity (2.59 ± 0.08 and 3.48 ± 0.13 µM/g liver/5 min formaldehyde production) checked.

Test concentrations with justification for top dose:

-S9: 2.44, 4.88, 9.76, 19.5, 39.1, 58.6 µg/mL
+S9: 2.44, 4.88, 9.76, 19.5, 39.1, 78.1, 117 µg/mL

The selection of the concentrations used in the main experiments was based on data from a preliminary cytotoxicity assay. Severe toxicity was observed at the seven highest dose-levels (with and without S9). Based on these results maximum concentrations of 58.6 and 117 µg/mL were selected for treatments in the absence and presence of S9 mix, respectively.

Vehicle / solvent:

Since no solvent vehicle was employed in this study the negative controls consisted of untreated cultures.

Controls

Untreated negative controls:

yes

Remarks:

untreated cells

Negative solvent / vehicle controls:

no

Remarks:

no vehicle used

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 : single
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10E6 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h at 37 °C

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 72 h
- Selection time (if incubation with a selective agent): 12 - 16 days
- Method used: agar
- Selection agent: Trifluorothymidine (final concentration 4.0 µg/mL)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 500,000 cells; after incubation, the plates are scored either manually or using a calibrated Artek Model 890 Automatic colony counter; the mutatioh frequency at each test point is calculated.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Percentage survival relative to the solvent controls is calculated for each treatment in a preliminary cytotoxicity experiment. Dose-levels giving a predicted 20% survival are estimated with and without S9. The estimated concentrations are chosen as the highest dose-levels for the mutation assays.

Evaluation criteria:

For a test substance to be considered mutagenic in this assay, it is required that:
(i) There is a two-fold (or more) increase in mutation frequency compared with the solvent control values, over two consecutive test substance treatment levels. If only the highest practicable dose-level (or the highest dose-level not to cause unacceptable toxicity) gives such an increase, then a single treatment-level will suffice.
(ii) The increases must be reproduced in an independent experiment.
(iii) There must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).

Statistics:

The results of the experiments were subjected to an Analysis of Variance, in which the contribution of experiment number and dose-level to the observed variation was examined.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 58.6 µg/mL -S9 and 177 µg/mL + S9

Vehicle controls validity:

not examined

Remarks:

no vehicle used

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The addition of the test substance solution did not have any obvious effect on the pH of the treatment medium.
- Data on osmolality: The addition of the test substance solution did not have any obvious effect on the osmolality of the treatment medium.
- Possibility of evaporation from medium: The test substance is not volatile.
- Water solubility: The test substance was soluble in complete medium at a concentration of 100 mg/mL. On the basis of these results, a maximum concentration of 10,000 µg/mL was selected for the cytotoxicity test.

RANGE-FINDING/SCREENING STUDIES
The test substance was assayed at a maximum concentration of 10,000 µg/mL and 8 lower dose levels spaced at two-fold intervals. Following treatment both in the absence and presence of S9 mix, severe toxicity was observed at the 7 highest dose levels, reducing total suspension growth to below the limit of detection. In the absence of S9 mix, at the next lower dose level (78.1 µg/mL) the total suspension growth value was reduced to 2% of the negative control. In the presence of S9 mix, no toxicity was observed at the next lower dose level (78.1 µg/mL). Maximum concentrations of 58.6 and 117 µg/mL were selected for the main study in the absence and presence of S9 mix, respectively.

TEST RESULTS:
For details please refer to Tables 1-4 and the attachment.

Any other information on results incl. tables

Table 1: Results of Experiment I without metabolic activation

Concentration [µg/mL]	Relative Plating Efficiency [%]	Total number of mutant colonies	Mean of mutation plate counts	S.D.
0	100	217	21.7	2.8
2.44	94	78	15.6	1.1
4.88	102	92	18.4	2.7
9.76	92	92	18.4	3.2
19.5	103	107	21.4	3
39.1	93	118	23.6	4
58.6	104	117	23.4	3
EMS, 2.5	71	710	142	13.1
EMS, 5.0	37	1039	207.8	11.8

 

Table 2: Results of Experiment I with metabolic activation

Concentration [µg/mL]	Relative Plating Efficiency [%]	Total number of mutant colonies	Mean of mutation plate counts	S.D.
0	100	149	14.9	2.4
2.44	98	104	20.8	1.3
4.88	100	112	22.4	4
9.76	101	57	11.4	1.5
19.5	96	67	13.4	2.1
39.1	97	57	11.4	2.7
78.1	97	67	13.4	2.1
117	90	65	13	3.4
DMBA, 2.0	64	229	45.8	5.7
DMBA, 3.0	52	236	59	5

 

Table 3: Results of Experiment II without metabolic activation

Concentration [µg/mL]	Relative Plating Efficiency [%]	Total number of mutant colonies	Mean of mutation plate counts	S.D.
0	100	178	17.8	3.7
2.44	99	93	18.6	3
4.88	95	76	15.2	1.8
9.76	99	79	15.8	3.1
19.5	94	75	15	2.3
39.1	95	83	16.6	3
58.6	115	76	15.2	2.2
EMS, 2.5	62	604	120.8	5.8
EMS, 5.0	27	588	117.6	13.6

 

Table 4: Results of Experiment II with metabolic activation

 

Concentration [µg/mL]	Relative Plating Efficiency [%]	Total number of mutant colonies	Mean of mutation plate counts	S.D.
0	100	164	16.4	4.2
2.44	85	72	14.4	1.8
4.88	95	87	17.4	2.7
9.76	83	68	13.6	2.1
19.5	114	114	22.8	3.1
39.1	92	75	15.0	3.0
78.1	99	126	25.2	5.0
117	-	-	-	-
DMBA, 2.0	114	164	82.0	7.1
DMBA, 3.0	122	205	102.5	14.8

'-': Not plated

Applicant's summary and conclusion

Conclusions:

In the present in vitro gene mutation study in mouse lymphoma (L5178Y) cells, the test substance did not induce gene mutations when tested at concentrations 117 and 58.6 µg/plate with and without metabolic activation, respectively. Therefore it is concluded that the test substance is not mutagenic in vitro in L5178Y mouse lymphoma cells, under the reported experimental conditions.