Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl laurate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-11-20 until 1993-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl laurate

EC Number:

236-942-6

EC Name:

Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl laurate

Cas Number:

13557-75-0

Molecular formula:

C18H32O6.Na

IUPAC Name:

sodium 2-{[2-(dodecanoyloxy)propanoyl]oxy}propanoate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch no. of test material: Unilever sample number S1986201

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Desiccated and refrigerated in the dark
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: 0.24 mg/mL in culture medium
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test chemical stock solution was prepraed by dissolving Pationic 138C in culture medium (with warming at 37 °C) to give 266.7 µg/mL. Then solution was membrane filter-sterilized and dilutions made in glass containers using culture medium. The test chemical solutions were protected from light and used within 1.5 hours of initial dissolution.
- Final dilution of a dissolved solid, stock liquid or gel: 266.7 µg/mL

FORM AS APPLIED IN THE TEST (if different from that of starting material) : in solution form for test; arrived as a beige paste.

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Healthy, nonsmoking volunteer
- Suitability of cells: not specified
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable: female
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable: blood was refrigerated during storage. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL heparinised blood into culture media, rocked continuously during incubation
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Hepes-buffered RPMI medium containing 20% (v/v) fetal calf serum and 50 ug/mL genatmycin. Phytohaemagglutinin (PHA, reagent grade) was included at a concentration of 37.5 uL per mL of culture to stimulate lymphocyte division.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

10.76, 15.37, 21.96, 31.37, 44.82, 64.03, 91.47, 130.7, 186.7, 266.7 µg/mL
TOP DOSE: At which a 50-80 % reduction in mitotic index occurred, a concentration close to the solubility limit in the treatment medium or 10 mM (or 5000 µg/mL), whichever was the lower.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

culture medium

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 20 hours
- Expression time (cells in growth medium): 3 hours with S9
- Fixation time (start of exposure up to fixation or harvest of cells): 37 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 per dose, with and without S9

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Lymphocytes were kept in fixative in the refrigerator at approx. 4 °C before slides were prepared. Cells were pelleted and resuspended in a minimal amoung of fresh fixative (milky suspension was the goal). Several drops of 45 % (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were dropped on to clean microscope slides which had been dipped in water. After the slides had dried, the cells were stained for 5 minutes in 4 % (v/v) filtered Giemsa stain in pH 6.8 buffer. Slides were rinsed, dried, and mounted with coverslips.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 metaphases per culture. Only cells with 44-46 chromosomes were considered acceptable for analysis of structural aberrations.

OTHER EXAMINATIONS:
- Determination of polyploidy: Any cell with more than 46 chromosomes were noted and recorded separately
- Determination of endoreplication: Any cell with more than 46 chromosomes were noted and recorded separately

Rationale for test conditions:

The human lymphocyte assay was valid if the following criteria were met:
1) The bionomial dispersion test demonstrated acceptable heterogeneity between replicate cultures
2) The proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the normal range
3) At least 160 cells out of an intended 200 were analysable at each treatment level
4) The positive control chemicals induced statistically significant increases in the number of cells with structural aberrations

Evaluation criteria:

The test chemical was to be considered as clearly positive in this assay if:
1) Statistically significant increases in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations
2) The proportion of aberrant cells at such data points exceeded the normal range.
Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations within the normal range or occurring only at very high or very toxic concentrations were likely to be concluded as equivocal. Cells with exchange aberrations or cells with greater than one aberration were to be considered of particular biological significance.

Statistics:

The proportion of cells in category 2 for each test treatment condition were compared with the proportion in negative controls using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human

Remarks:

Single female healthy non-smoking volunteer

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

same as vehicle control

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: 0.24 mg/mL in test medium
- Definition of acceptable cells for analysis: 44-46 chromosomes

HISTORICAL CONTROL DATA
- Positive historical control data: not given
- Negative (solvent/vehicle) historical control data - mean (range):
- structural aberrations including gaps: 2.2 (0-8) without S9; 2.5 (0-8) with S9
- structural aberrations excluding gaps: 0.5 (0-3) without S9; 0.9 (0-4) with S9
- numerical aberrations: 0.6 (0-3) without S9; 0.7 (0-3) with S9

Any other information on results incl. tables

VALIDITY OF STUDY

The data confirm that:

1) No evidence of significant heterogeneity between replicate cultures was obtained in the binomial dispersion test

2) The proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the normal range

3) At least 160 cells out of an intended 200 were analysed at each treatment level

4) The positive control chemicals NQO and CPA induced statistically significant increases in the number of cells with structural aberrations.

A small increase in cells with numerical aberrations was observed in one treated culture but only in one of the pair of replicates and was considered spurious.

Applicant's summary and conclusion

Conclusions:

Sodium lauroyl lactylate (Pationic 138C) did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of solubility in both the absence and presence of S-9.

Executive summary:

In a test following OECD guideline 473 (in vitro mammalian chromosome aberration test), human lymphocyte cultures treated with sodium lauroyl lactylate (Pationic 138C) in both the absence and presence of S9 had frequencies of cells with aberrations which were similar to and not significantly different from those in concurrent solvent controls. Numbers of aberrant cells fell within historical negative control ranges under all treatment conditions. Therefore, in this test system sodium lauroyl lactylate is not identified as genotoxic.