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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid testing guidelines, therefore it is considered relevant, adequate and reliable for classification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - 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:
Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts
EC Number:
290-836-4
EC Name:
Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts
Cas Number:
90268-36-3
Molecular formula:
C12 - C18 C16 H30 O7 S 2Na - C22 H42 O7 S 2Na
IUPAC Name:
Butanedioic acid, sulfo-, 1-C12-18 (even numbered)-alkyl esters, disodium salts
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts; Disodium Lauryl Sulfosuccinate
- Physical state: White powder
- Analytical purity: > 95% (correction factor: 1.05)
- Impurities (identity and concentrations): See confidential details
- Composition of test material, percentage of components: See confidential details
- Purity test date: 2012-07-03
- Lot/batch No.: 0008249628
- Expiration date of the lot/batch: March 20, 2014
- Stability under test conditions: stable
- Storage condition of test material: At+10°to+25°C
- Other: manufacturer/supplier: BASF Personal Care and Nutrition GmbH

Method

Target gene:
hprt locus at the X-chromosome
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
*V79 cells were maintained in Dulbecco's modified Eagle-Medium supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS. Cultures were incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2.
*For subculturing, a trypsin (0.05%)-EDTA (ethylenediamine¬tetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.
*Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).
- Properly maintained: yes.
- Periodically checked for Mycoplasma contamination: yes, by using the HOECHST stain 33258.
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes. The spontaneous mutation rate was continuously monitored.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 19.53, 39.06, 78.13, 156.3, 312.5, 625, and 1250 µg test item/mL
Main test without S9-mix: 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL
Main test with S9-mix: 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
aqua ad iniectabilia
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate in DMSO
Remarks:
600 and 700 µg/mL, without S9-mix
Positive controls:
yes
Positive control substance:
other: 9,10-dimethyl-1,2-benzanthracene in DMSO
Remarks:
20 and 30 µg/mL with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
The enzyme hypoxanthine guanine phosphoribosyl transferase (hprt) catalyses phosphorylation of purines in one of the purine salvage pathways. The selective agent used in this assay, 6-thioguanine (6-TG), is also a substrate for this enzyme and cells that retain the functional hprt enzyme are susceptible to the cytotoxic effects of 6-TG. Forward mutations that result in the loss of the functional hprt gene render cells resistant to 6-TG. These mutant cells can be quantitated after an expression period by cloning in culture medium supplemented with 6-TG, the selective agent.

DURATION
- Preincubation period: 24 hours
- Exposure duration: Without S9-mix:4 hours (1st experiment) or 24 hours (2nd experiment); With S9-mix: 4 hours
- Expression time (cells in growth medium): until day 8 with one subcultivation on day 5
- Selection time (if incubation with a selection agent): about 8 days (plating efficiency plates: cytotoxicity test) or 12 days (6-thioguanine plates: mutagenicity test).

SELECTION AGENT (mutation assays): 6-thioguanine (10 µg/mL);
STAIN (cytogenetic assays): After about 8 days, the cells were fixed and stained with methylene blue in ethanol. The colonies were then counted.

NUMBER OF REPLICATIONS:
cytotoxicity: triplicate
mutagenicity: for selection of mutants 5 replicate plates; for the estimation of plating efficiencies (PE) 3 replicate plates.

DETERMINATION OF CYTOTOXICITY
- Method: other: relative plating efficiency is determined for each dose to obtain an accurate measure of the toxic effect of the chemical.
Evaluation criteria:
So far no satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments such as those performed here (see UKEMS guidelines for discussion). Following pre¬determined descriptive criteria are used for interpretation of the results:
-lf in both independent experiments solvent and positive controls show results within the norm and if the test compound does not increase the mutation, or if the mutation frequency is always lower than 40 x 10-6 and if at least 1 000 000 cells per condition have been evaluated, the compound is considered as negative in the test.
-In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold solvent control and at least 40 x 10-6 both in the presence and/or absence of S9 mix, the compound is considered as positive in the test.
Equivocal results, if applicable are clarified by further testing, in agreement with Sponsor and Study Monitor.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No changes in the pH values in the medium were noted.
- Effects of osmolality: No relevant changes in osmolality of the formulations were noted.

RANGE-FINDING/SCREENING STUDIES:
- In the preliminary study cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 39.06 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 156.3 µg/mL in the presence of metabolic activation.
- The next higher concentrations resulted in complete cytotoxicity in the preliminary experiment. In addition, the number of mutants was also already considerable decreased in the main experiments at the highest employed concentrations pointing to general pronounced cytotoxicity. Finding a higher concentration with viable and evaluable cells/mutants was therefore not considered realistic.
- In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 106 survivors in non-activation solvent controls and 6 to 46 per 106 survivors in S9 activation solvent controls.
The mutation frequency of the cultures treated with concentrations of 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL culture medium without metabolic activation ranged from 3.78 to 15.74 x 10-6 clonable cells. These results are within the normal range of the vehicle controls.
The mutation frequency of the cultures treated with concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL culture mediumwith metabolic activation ranged from 4.44 to 13.04 x 10-6 clonable cells. These results are within the normal range of the vehicle controls.
The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 475.00 to 780.00 x 10-6 clonable cells in the case of EMS and ranging from 530.71 to 855.00 x 10-6 clonable cells in the case of DMBA, indicating the validity of this test system.
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA.



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 with metabolic activation
negative without metabolic activation

Under the present test conditions, the test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

The test item was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item.  The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary study test item concentrations of 19.53, 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/mL medium were employed . Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 39.06 µg test item/mL was employed as the top concentration for the mutagenicity tests in the absence and 156.3 µg/mL in the presence of metabolic activation.

Main study

Five concentrations 2.44, 4.88, 9.77, 19.53 or 39.06 and 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL were selected for the experiments without and with metabolic activation, respectively.

Cytotoxicity

In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2)was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.

Experiments without metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 14.35 and 16.67 x 10-6clonable cells. Hence, the vehicle controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL culture medium ranged from 4.44 to 13.04 x 106clonable cells. These results are within the normal range of the vehicle controls.

Experiments with metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 17.87 and 16.05 x 10-6clonable cells. Hence, the vehicle controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL culture medium ranged from 3.78 to 15.74 x 106clonable cells. These results are within the normal range of the vehicle controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 475.00 to 780.00 x 10-6clonable cells in the case of EMS and ranging from 530.71 to 855.00 x 10-6clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6clonable cells for EMS and 130.0 to 2693.3 x 10-6clonable cells for DMBA.

Under the present test conditions, the test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.