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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:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 476, EC B.17 and US EPA OPPTS 870.5300 and in acordance with the Principles of Good Laboratory Practice (GLP).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1-(Decyloxy)-2-(1-methylpropyl)-4-(triphenylmethyl) benzene
- Physical State: Brown viscous liquid to semi solid
- Analytical Purity: 92.6%
- Lot/Batch No.: lot ZA-03012014M

Method

Target gene:
The in vitro mammalian cell gene mutation tests is used to detect gene mutations induced by chemical substances. Suitable cell lines include L5178Y mouse lymphoma cells, the CHO, AS52 and V79 lines of Chinese hamster cells, and TK6 huan lymphoblastoid cells. In these cell lines the most commonly-used genetic enpoints measure mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT), and a transgene of xanthineguanine phosphoribosyl transferase (XPRT). The TK, HPRT, and XPRT mutation tests detect different spectra of genetic events. The autosomal location of TK and XPRT allows the detection of genetic events (e.g. large deletions) not detected at the HPRT locus on the X-chromosomes.
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 medium (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, 0.25 µg/ml; GIBCO), and an additional 2 mM L-glutamine (GIBCO)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 12545
Test concentrations with justification for top dose:
Preliminary Toxicity Assay - 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, and 150.0 µg/ml
Initial Mutagenicity Assay - 1.5, 4.74, 15.0, 47.4, and 150 µg/ml
Confirmatory Gene Mutation Assay - 1.5, 4.74, 15.0, 47.4, and 150 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 1% Ethanol
- Justification for choice of solvent/vehicle: Test material solubiility
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
The solvent ethanol selected for dissolving the test material was used as the negative control treatment.
True negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: 20-methylcholantherene
Remarks:
Ethyl methanesulfonate (EMS, CAS No. 62-50-0) at 621ug/ml and 20-methylcholanthrene (20-MCA, CAS No. 56-49-5) at 4 and 8 ug/ml.
Details on test system and experimental conditions:
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge National Laboratory, Oak Ridge, Tennessee, was used in this study. The CHO-K1-BH4 cell line was selected as the test system for this study because it is sensitive to mutagens, has a low background mutant frequency, and is readily available. Stock cultures were stored at about -80°C or below. The cultures were periodically checked for mycoplasma contamination (American Type Culture Collection, Manassas, Virginia). The cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of approximately 5% CO2 in air at 37C in a humidified incubator.

The test material was found to be soluble in ethanol (ethanol; CAS No. 64-17-5) up to 62.65 mg/ml. Therefore, ethanol was selected as the solvent used to dissolve the test material and was used as the vehicle control treatment. Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was used as the positive control for the non-activation system (without S9 factor) at a final concentration of 621 g/ml. The positive control for assays performed with S9 (activation system) was 20-methylcholanthrene (20-MCA, CAS No. 56-49-5) at concentrations of 4 and 8 g/ml. The dose levels of EMS and 20-MCA were based upon our unpublished findings.

All test material solutions were prepared fresh on the day of treatment and used within one hour of preparation. The test material was first dissolved in ethanol and further diluted (1:100) with the treatment medium to obtain the desired concentrations. This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et al, 1981). EMS was dissolved in treatment medium. 20-MCA was dissolved first in dimethyl sulfoxide and further diluted in the culture medium. All dosing units were expressed in µg/ml.

Cells in logarithmic growth phase were trypsinized and placed in medium containing 5% serum at a standard density of 3.0 x 106 cells/T-75 flask approximately 24 hours prior to treatment. At the time of treatment, the culture medium was replaced with serum-free medium, S9 mix (when applicable), and the test chemical, the vehicle control, or the positive control chemical. The cells were treated for approximately 4 hours at 37°C and the exposure was terminated by washing the cells with phosphate buffered saline (Ca++ and Mg++ free).

All test cultures were identified using self adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

The selected concentrations of the test material in the stock dosing solutions used for treatment in the initial gene mutation assay (Assay B1) were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in acetonitrile and analyzed by high performance liquid chromatography with ultraviolet detection (HPLC/UV).

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. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a “mix” (O’Neill et al., 1982). The S9 mix consisted of the following co-factors: 10 mM MgCl2•6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the culture medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the culture medium is 1/5th of the concentrations stated above.

Toxicity Assay - The cytotoxicity of the test material was assessed by determining the ability of the treated cells to form colonies. This assay was conducted for selecting concentrations of the test material to be used in the gene mutation assay. Cells were seeded into T-25 flasks (1.0 x 106 cells/flask) approximately 24 hours prior to treatment. Treatment was for approximately 4 hours with various concentrations of the test material with one replicate per dose in the presence and absence of S9. After termination of treatment, the cells were trypsinized and replated at a density of 200 cells/dish into 60 mm dishes (3/dose) and the dishes incubated for 7 days to allow colony formation. The colonies were then fixed/stained with methanol/crystal violet. The number of colonies/dish was counted and the mean colonies/treatment were expressed relative to the vehicle control value. The highest dose level of the test material (150 µg/ml) was limited by the solubility of the test material in the treatment medium. The other concentrations tested were separated by a factor 2.

Gene Mutation Assay - Based upon the toxicity assay, precipitating dose levels in the absence and presence of S9 were selected as the top concentrations for each system. At least four additional concentrations of the test material, the lowest of which was expected to give cell survival comparable to vehicle control, were also tested in the gene mutation assay. The concentrations were usually spaced apart by a factor of 3.2. The cytotoxicity of the selected concentrations was determined concurrent with the gene mutation assay.Each dose level was set up in duplicate from the time of treatment until the completion of the assay. The cultures were trypsinized at the end of the treatment and replated at a density of 1 x 106 cells/100 mm dish (at least two dishes/replicate) for phenotypic expression (O'Neill et al., 1977a; O'Neill and Hsie, 1979). In addition, 200 cells/60 mm dish (three dishes/replicate) were also plated to determine the toxicity and incubated for approximately 7 days to permit colony formation. During the phenotypic expression period (8 days), cells in the larger petri dishes were subcultured every 2-3 days and plated (at least two dishes/replicate) at a density of about 1 x 106 cells/100 mm petri dish. At each subculture, cells from various dishes within each replicate were pooled prior to replating. At the end of the expression period, the cultures were trypsinized and plated at a density of 2 x 105 cells/100 mm dish (a total of 10 dishes/treatment) in the selection media (Ham's F-12 without hypoxanthine and with 6-thioguanine) for the determination of Hgprt- mutants and 200 cells/60 mm dish (three dishes/treatment) in Ham's F-12 medium without hypoxanthine for determination of cloning efficiency. The dishes were incubated for about 8 days and the colonies were fixed/stained with methanol/crystal violet. The mutant frequency (expressed as mutants per 106 clonable cells) for each replicate were calculated by the following formula (Kirkland, 1989):
MF = K x (m / c)

Where:
K = Pc x 106 / Pm

Pc = the number of cells plated for the survival plates
Pm = the number of cells plated for the mutation plates
c = the mean colonies per plate for survival plates for each replicate
m = the mean colonies per plate for mutation plates for each replicate


Evaluation criteria:
For an assay to be acceptable, the mutant frequency in positive controls should have been significantly higher than the vehicle controls. An additional criterion was that the mutant frequency in the vehicle 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 vehicle 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 mutant 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 vehicle control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the vehicle 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 applicable
Positive controls validity:
valid
Additional information on results:
pH and Osmolality
The pH and osmolality of treatment medium containing approximately 157 µg/ml of the test material and medium containing 1% ethanol were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays (Thilagar et al., 1984; Galloway et al., 1985; Cifone, 1985). There was no appreciable change in either the pH or osmolality at this concentration as compared to the culture medium with solvent alone (culture medium with the test material, pH = 7.36, osmolality = 492 mOsm/kgH2O; culture medium with 1% ethanol, pH = 7.32, osmolality = 492 mOsm/kgH2O).

Assay A1 – Preliminary Toxicity Assay
In a preliminary toxicity assay, the test material was tested at concentrations of 0 (vehicle control), 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, and 150.0 µg/ml in the absence and presence of an externally supplied metabolic activation system (S9). The highest three concentrations (37.5, 75.0, and 150.0 µg/ml) precipitated in the treatment medium. The treated cultures in the absence and presence of S9 activation showed little to no toxicity with the relative cell survival (RCS) values ranging from 112.9 to 146.5% in the absence of S9 and 90.2 to 165.0% in the presence of S9. Based upon the results of this assay, target concentrations of 0 (vehicle control) 1.5, 4.74, 15.0, 47.4, and 150 µg/ml of the test material were selected for the initial gene mutation assay in the absence of S9 and presence of S9.

Assay B1 – Initial Mutagenicity Assay
In the initial mutagenicity assay (Assay B1), the highest two concentrations (47.4 and 150 µg/ml) precipitated in the treatment medium. In the absence of S9, little to no toxicity was observed with RCS values ranging from 103.1 to 127.4%. In the presence of S9, little to no toxicity was observed with RCS values ranging from 103.5 to 139.6%. The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 were not significantly different from the concurrent vehicle control values. All mutant frequencies were within a reasonable range of historical background values.

Assay C1 – Confirmatory Mutagenicity Assay
Based upon the previous results, targeted concentrations of 0 (vehicle control), 1.5, 4.74, 15, 47.4, and 150 µg/ml in the absence and presence of S9 were selected for the confirmatory gene mutation assay. In this assay, the highest two concentrations precipitated in the treatment medium. There was little to no toxicity observed, as indicated by RCS values, in the absence of S9 (103.4 to 131.3%). In the presence of S9, RCS values indicated little to no toxicity with RCS values ranging from 76.9 to 122.5%. The mutant frequencies observed in cultures treated with the test material in the absence of S9 and presence of S9 were not significantly different from the concurrent vehicle control values and were within the range of the laboratory historical background.
In both the initial and confirmatory mutagenicity assays, the positive control chemicals induced significant increases in mutant frequencies and this data confirmed the adequacy of the experimental conditions for detecting induced mutations.
The analytically determined concentrations of the test material in the stock dosing solutions in Assay B1 ranged from 87.5 to 94.7% of target and verified that concentrations used for treatment were within acceptable range.
Remarks on result:
other: other: CHO-K1-BH4
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results of the in vitro Chinese Hamster Ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay with ACCUTRACE™ S10 Fuel Marker indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.
Executive summary:

Accutrace™ S10 Fuel Marker (1-(Decyloxy)-2-(1-methylpropyl)-4-(triphenylmethyl) benzene) 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 1.5 to 150 µg/ml in the absence and presence of S9. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The analytically-determined concentrations of Accutrace™ S10 Fuel Marker in the dose preparations ranged from 87.5 to 94.7% of the targeted values. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Vehicle control cultures were treated with the solvent used to dissolve the test material (i.e. ethanol). There were no statistically significant treatment-related increases in the mutant frequency in the test material-treated cultures compared to the vehicle control cultures in either the absence or presence of S9. The results of the CHO/HGPRT forward gene mutation assay with ACCUTRACE™ S10 Fuel Marker indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.