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Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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 coloniesMean of mutation plate countsS.D.
010021721.72.8
2.44947815.61.1
4.881029218.42.7
9.76929218.43.2
19.510310721.43
39.19311823.64
58.610411723.43
EMS, 2.57171014213.1
EMS, 5.0371039207.811.8

 


Table 2: Results of Experiment I with metabolic activation


















































































Concentration [µg/mL]Relative Plating Efficiency [%]Total number of mutant coloniesMean of mutation plate countsS.D.
010014914.92.4
2.449810420.81.3
4.8810011222.44
9.761015711.41.5
19.5966713.42.1
39.1975711.42.7
78.1976713.42.1
1179065133.4
DMBA, 2.06422945.85.7
DMBA, 3.052236595

 


Table 3: Results of Experiment II without metabolic activation


















































































Concentration [µg/mL]Relative Plating Efficiency [%]Total number of mutant coloniesMean of mutation plate countsS.D.
     
010017817.83.7
2.44999318.63
4.88957615.21.8
9.76997915.83.1
19.59475152.3
39.1958316.63
58.61157615.22.2
EMS, 2.562604120.85.8
EMS, 5.027588117.613.6

 


Table 4: Results of Experiment II with metabolic activation


 


















































































Concentration [µg/mL]Relative Plating Efficiency [%]Total number of mutant coloniesMean of mutation plate countsS.D.
010016416.44.2
2.44857214.41.8
4.88958717.42.7
9.76836813.62.1
19.511411422.83.1
39.1927515.03.0
78.19912625.25.0
117----
DMBA, 2.011416482.07.1
DMBA, 3.0122205102.514.8

'-': 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.