Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 06,2012 to August 03,2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant with international guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts

EC Number:

270-307-4

EC Name:

Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts

Cas Number:

68424-50-0

Molecular formula:

UVCB

IUPAC Name:

Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts

Test material form:

liquid: viscous

Details on test material:

- Name of test material: Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts
- Molecular formula: UVCB
- Physical state: clear yellowish brown gel
- Analytical purity:
- Purity test date:
- Lot/batch No.:CH159604/01
- Expiration date of the lot/batch:08-May-2013
- Storage condition of test material:the storage conditions for the test item will be room temperature.

Method

Target gene:

no data

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Main Assay I:2500, 1250, 625, 313 and 156 µg/plate
Main assay II2500, 791, 250, 79.1 and 25.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:Dimethylsulfoxide (DMSO) ,sterile water for injection
- Justification for choice of solvent/vehicle:These solvents were selected since they are compatible with the survival of the bacteria and the S9 metabolic activity. A slightly opaque solution, suitable for treatment but not for serial dilution, was obtained at 25 mg/mL in DMSO. This result permitted a maximum concentration of 2500 µg/plate to be used in the toxicity test.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

wothout S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar
Nutrient Broth: Oxoid Nutrient Broth No 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
Nutrient Agar: Oxoid Nutrient Broth No 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved.
The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
Minimal Agar: Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use 10 mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 mL).

DURATION
- Preincubation period:72h at 37°C
For the pre-incubation method, due to DMSO toxicity, 50 µL of the test item solution is used in the preparation of each plate.

NUMBER OF REPLICATIONS:Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.

NUMBER OF CELLS EVALUATED:scoring was effected by counting the number of revertant colonies on each plate.
100 - 500 million for each strain.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic relative total growth

OTHER EXAMINATIONS:
- Sterility check: The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions

OTHER:
The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.

The overlay mixture was composed as follows:
(i) Overlay agar (held at 45°C) 2 mL
(ii) Test or control item solution 0.1 mL
(iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 mL
(iv) Bacterial suspension 0.1 mL
The second experiment was performed using a pre-incubation method.
The components were added in turn to an empty test-tube:
(i) Bacterial suspension 0.1 mL
(ii) Test item solution or control item solution 0.05 mL
(iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 mL

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

i)The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as:
y = a + bx
where
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
ii)The regression line does not include the untreated control data, but includes the solvent control data.
iii)Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:No precipitation of the test item was observed at the end of the incubation period at any concentration. However, agar plates treated at the highest dose level showed a slight opacity.
- Other confounding effects:
The negative control values for TA1535 in the presence of S9 metabolism were slightly higher than the upper confidence limit. However, the results obtained were lower than the maximum value of the historical control range and consistent with data generated by scientific community; hence they were considered acceptable.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that the test item TALLOELFETTSAEURE-C15+-ALKYLESTER, SULFONIERT, NA-SALZ does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item TALLOELFETTSAEURE-C15+-ALKYLESTER, SULFONIERT, NA-SALZ  was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

The test item was used as a solution in dimethylsulfoxide (DMSO).

The test item TALLOELFETTSAEURE-C15+-ALKYLESTER, SULFONIERT, NA-SALZ was assayed in the toxicity test at the maximum dose level of 2500 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 791, 250, 79.1 and 25.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. However, agar plates treated at the highest dose level showed a slight opacity. No toxicity was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

In Main Assay I, using the plate incorporation method, the test item was assayed at the maximum dose level of 2500 µg/plate and at four lower dose levels spaced by two-fold dilutions, 1250, 625, 313 and 156 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. However, a slight opacity of agar plates was observed at the highest dose level. No toxicity was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

As no increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II.

In the pre-incubation method, due to DMSO toxicity, 50 µL of the test item solution, instead of 100 µL, is used in the preparation of each plate. Therefore, the test item was assayed at the maximum concentration of 1250 µg/plate and at four lower dose levels spaced by two fold dilutions 625, 313, 156 and 78.1 µg/plate.

No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item TALLOELFETTSAEURE-C15+-ALKYLESTER, SULFONIERT, NA-SALZ does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.