Reaction mass of sodium 1-methoxy-1-oxohexadecane-2-sulphonate and sodium methyl 2-sulphooctadecanoate

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:

19 February - 22 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

In the second mutagenicity test in the absence of S9-mix, only five dose levels could be analyzed for expression of the TK mutation.
Evaluation: Too many dose levels showed a toxic response to the treatment. The two highest tested dose levels showed appropriate toxicity: a RTG 75 and 89%, the testing of lower dose levels would have given limited additional information; therefore the use of only five dose levels has no effect on the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1-methoxy-1-oxohexadecane-2-sulphonate

EC Number:

223-676-0

EC Name:

Sodium 1-methoxy-1-oxohexadecane-2-sulphonate

Cas Number:

4016-24-4

Molecular formula:

There are different notations of the molecular formula, dependent on whether it is depicted as protonated or not: C17H34O5S.Na C17H33NaO5S C14H29CH(CO2CH3)SO3Na CH3(CH2)13CH(SO3Na)COOCH3

IUPAC Name:

sodium 1-methoxy-1-oxohexadecane-2-sulfonate

Constituent 2

Reference substance name:

Sodium methyl 2-sulphooctadecanoate

EC Number:

223-770-1

EC Name:

Sodium methyl 2-sulphooctadecanoate

Cas Number:

4062-78-6

Molecular formula:

There are different notations of the molecular formula, dependent on whether it is depicted as protonated or not: C19H38O5S.Na or C19H37O5S.Na CH3(CH2)15CH(SO3Na)COOCH3

IUPAC Name:

sodium methyl 2-sulphooctadecanoate

Test material form:

solid: flakes

Details on test material:

- Physical state: pale yellow flakes
- Storage condition of test material: In refrigerator (2-8 ºC) in the dark

Method

Target gene:

thymidine kinase (TK) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

First mutagenicity test:
Without S9-mix: 0.5, 1, 5, 10, 15, 30, 40, and 50 μg/ml exposure medium.
With 8% (v/v) S9-mix: 1, 10, 33, 66, 100, 110, 115 and 120 μg/ml exposure medium.

Second mutagenicity study:
Without S9-mix: 0.3, 3, 30, 50, and 55 µg/ml exposure medium.
With 12% (v/v) S9-mix: 1, 10, 33, 100, 110, 115, 120, and 130 μg/ml exposure medium.

Vehicle / solvent:

DMSO
The final concentration of the solvent in the exposure medium was 0.8% (v/v)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

for the 3 and 24 hrs treatment -S9 mix; at 15 and 5 µg/ml in DMSO

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

for + S9-mix treatments; at 7.5 µg/ml in Hanks' balanced salt solution (- Ca and -Mg)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for
1 day in R10 medium containing 10-4 M hypoxanthine, 2 x 10-7 M aminopterine and 1.6 x 10-5 M thymidine to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.
- Exposure duration: 3 and 24 hrs (+ and - 8% (v/v) S9 mix
- Expression time (cells in growth medium): 2 days after the end of the treatment
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): trifluoro thymidine (TFT)
STAIN (for cytogenetic assays): 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED: 8 x 10^6 cells (10^6 cells/ml for 3 hours treatment) or 5 x 10^6 cells (1.25 x 10^5 cells/ml for 24 hours treatment)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (106) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 x 10-6 and ≤ 170 x 10-6.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 x 10-6, and for CP not below 700 x 10-6.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more then MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility and precipitation: MIZULAN FL-80 precipitated in the exposure medium at concentrations of 333 μg/ml and above. MIZULAN FL-80 was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 1000 μg/ml.


RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 10 to 1000 µg/ml in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
In the absence of S9-mix, the relative suspension growth was 46% at the test substance concentration of 33 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 100 μg/ml and above.
In the presence of S9-mix, the relative suspension growth was 48% at the test substance concentration of 100 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 333 μg/ml and above.
In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 10 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 33 μg/ml and above.


COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. The observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First test: In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 89% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 89% compared to the total growth of the solvent controls.
Second test: In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 90% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 82% compared to the total growth of the solvent controls.

Applicant's summary and conclusion

Conclusions:

In the absence of S9-mix, MIZULAN FL-80 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.
In the presence of S9-mix, MIZULAN FL-80 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.
In conclusion, MIZULAN FL-80 is not mutagenic in the TK mutation test system under the experimental conditions described in this report.