Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been conducted according to the following guidelines - OECD, Guideline 476 (1997) and EC, B.17 (2000) and in accordance to Principles of GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

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
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Methyl Polyhydroxymethyl Stearate (Imperial Monomer)
- Molecular formula (if other than submission substance): UVCB- not applicable
- Molecular weight (if other than submission substance): UVCB- MW of primary components range from 270- 389 g/mole
- Smiles notation (if other than submission substance): UVCB- not applicable
- InChl (if other than submission substance): not specified in the report
- Structural formula attached as image file (if other than submission substance): UVCB- not applicable
- Substance type: Mixture of functionalized fatty acid methyl esters (FAMEs), which are derived from a variety of natural seed oils.
- Physical state: Waxy solid at room temperature
- Analytical purity: > 95.2 % as summed concentration of five primary components
- Impurities (identity and concentrations): water, approx. 0.4%
- Composition of test material, percentage of components:
Methyl Palmitate: 9.68 %
Methyl Stearate: 17.8 %
Methyl Hydroxymethyl Stearate: 38.6 %
Methyl Dihydroxymethyl Stearate: 26.7 %
Methyl Trihydroxymethyl Stearate: 2.37 %
- Isomers composition: The balance of unassigned composition can be attributed to both minor isomers of the primary components, as well as trace components resulting from physical/chemical processing of the unrefined natural oil feedstock.
- Purity test date: 22 August 2005
- Lot/batch No.: 200500200-25-4
- Characterisation expiration date: 07 September 2007
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark under nitrogen

Method

Target gene:
Induction of forward mutations in the Hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) gene in Chinese hamster overary (CHO) cells.
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells were routinely maintained in Ham's F-12 nutrient mix (GIBCO, Grand Island, New York) supplemented with 5% (v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum (GIBCO), antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 25 μg/ml; GIBCO) and an additional 2 mM L-glutamine (GIBCO). The selection medium used for the detection of HGPRT- mutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 μM 6-thioguanine (GIBCO) and 5% serum and the above mentioned antibiotics.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified in the report
- Periodically "cleansed" against high spontaneous background: not specified in the report
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats were purchased from a commercial source and stored at approximately -80°C or below.
Test concentrations with justification for top dose:
Preliminary toxicity assay - 0 (solvent control), 0.5, 1.67, 5, 16.7, 50, 167, 500, 1667, and 5000 μg/ml in the presence and absence of S9 metabolic activation system
Based upon the results of this assay, concentration levels of 0 (solvent control) 0.5, 5, 50, 500, and 5000 μg/ml of the test material were selected for the initial gene mutation assay in the absence of S9 and 0 (solvent control) 0.5, 5, 10, 20, 30, 40, 50, 60, and 100 μg/ml in the presence of S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The test material was first dissolved in ethyl alcohol and further diluted (1:100) with the treatment medium to obtain the desired concentrations
- Justification for choice of solvent/vehicle: solubility and non-cytogenic/mutagenic
Controls
Negative solvent / vehicle controls:
yes
Remarks:
ethyl alcohol
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
The positive control for assays performed with S9 (activation system) was 20-methylcholanthrene (20-MCA, CAS No. 56-49-5) at a concentration of 4 and 8 mg/ml

Migrated to IUCLID6: Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was used as the positive cont
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: Cells in logarithmic growth phase were trypsinized and placed in medium containing 5% serum at a standard density of 3.0 x 10 6 cells/T-75 flask approximately 24 hours prior to treatment.
- Exposure duration: 6-10 days
- Expression time (cells in growth medium): 4 hours
- Selection time (if incubation with a selection agent): not specified in the report
- Fixation time (start of exposure up to fixation or harvest of cells): 6-10 days


SELECTION AGENT (mutation assays): not specified in the report
SPINDLE INHIBITOR (cytogenetic assays): not specified in the report
STAIN (for cytogenetic assays): cyrstal violet


NUMBER OF REPLICATIONS: At least two dishes/replicate - Each dose level was set up in duplicate from the time of treatment until the completion of the assay.


NUMBER OF CELLS EVALUATED: not specified in the report


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


OTHER EXAMINATIONS:
- Determination of polyploidy: not specified in the report
- Determination of endoreplication: not specified in the report
Evaluation criteria:
For an assay to be acceptable, the mutant frequency in positive controls should have been significantly higher than the solvent controls. An additional criteria, was that the mutant frequency in the solvent controls should have been within reasonable limits of the laboratory historical control values and literature values. The test chemical was considered positive if it induced a statistically significant, dose related, reproducible increase in mutant frequency. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the solvent controls.
Statistics:
The frequency of mutants per 106 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutation frequency variance. The actual plate counts are assumed to follow a Poisson distribution therefore the mean plate count was used as an estimate of variance (Kirkland, 1989).

If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). An additional comparison of the positive control to the solvent control (alpha = 0.05) was conducted using a linear contrast statement. Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the solvent control yielded significant differences.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified in the report
- Effects of osmolality: not specified in the report
- Evaporation from medium: not specified in the report
- Water solubility: not specified in the report
- Precipitation: At 50 μg/ml and higher, the treatment medium was cloudy indicating precipitation of the test material.
- Other confounding effects: not specified in the report


RANGE-FINDING/SCREENING STUDIES: not specified in the report


COMPARISON WITH HISTORICAL CONTROL DATA: yes


ADDITIONAL INFORMATION ON CYTOTOXICITY: not specified in the report

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results of the in vitro Chinese hamster ovary cell/hypoxanthine-quanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay with methyl polyhydroxymethyl stearate (imperial monomer) indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the presence or absence of an externally supplied metabolic activation (S9) system.
Executive summary:

Methyl polyhydroxymethyl stearate (imperial monomer) was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 0.5 to 5000 μg/ml in the absence of S9 and from 0.5 to 100 μg/ml in the presence of S9. The highest concentration in the absence of S9 was based on the 5000 mg/ml limit dose for the assay system. In the presence of S9, the highest

concentration was based on the cytotoxicity observed in the initial toxicity assay. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays without S9 and 20-methylcholanthrene for assays with S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e. ethyl alcohol). The mutant frequencies observed in cultures treated with the test material in the absence or presence of S9 were not statistical significantly different from the concurrent solvent control values. The results of the in vitro Chinese hamster ovary cell/hypoxanthine-quanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay with methyl polyhydroxymethyl stearate (imperial monomer) indicate that under the conditions of this study, the test article was non-mutagenic

when evaluated in the presence or absence of an externally supplied metabolic activation (S9) system.

Categories Display