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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Feb - May 2012 (specific dates not provided in the report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
ANGUS Chemical Company, a wholly owned subsidiary of The Dow Chemical Company, Sterlington, Louisiana (Lot# ZD0431LAH8) supplied the test material. The purity of the test material was determined to be 99.9% area by gas chromatography with identification by nuclear magnetic resonance and gas chromatography mass spectrometry.

Method

Target gene:
HGPRT
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge National Laboratory, Oak Ridge, Tennessee, was used in this study. 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.
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
0, 46.9, 93.9, 187.8, 375.5 and 751 ug/ml with and without metabolic activation
Vehicle / solvent:
The solvent used to dissolve the test material (i.e., distilled water (CAS No. 7732-18-5)) was used as the solvent control treatment.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Ethyl methanesulfonate (EMS) was used as the positive control for the non-activation systemat a final concentration of 621 μg/ml. The positive control for assays performed with S9 was 20-methylcholanthrene (20-MCA) at concentrations of 4 and 8 μg/ml.
Details on test system and experimental conditions:
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, 0.25 μg/ml; GIBCO), and an additional 2 mM L-glutamine (GIBCO). The selection medium used for the detection of HGPRTmutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 μM 6-thioguanine (GIBCO), 5% serum, and the above-mentioned antibiotics.

Treatment Procedure
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. At the time of treatment, the culture medium was replaced with serum-free medium, S9 mix (when applicable) and the test chemical, the solvent 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). Volatility of the test material was assessed prior to treatment and deemed minimal, however, the caps of the flasks used for treatment were tightly sealed to ensure all test material remained in the treatment flask during the 4 hour treatment.

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

Preparation of the Treatment Solution
All test material solutions were prepared fresh on the day of treatment and used within two hours of preparation. The test material was first dissolved in distilled water and further diluted (1:10) with the treatment medium to obtain the desired concentrations. 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.

Analytical Verification of Dosing Solutions
The selected concentrations of the test material in the stock dosing solutions used for treatment in the initial mutagenicity assay (Assay B1) were verified by the Analytical Chemistry Laboratory, Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in an appropriate solvent and analyzed by gas chromatography with mass spectrometry detection (GC/MS).

In Vitro Metabolic Activation
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 10(6) 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 factor. After termination of treatment, the cells were trypsinized and replated at a density of 200 cells/dish into 60 mm dishes (three/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 were counted and the mean colonies/treatment were expressed relative to the solvent control value. The highest concentration tested was the guideline limit of 10 mM for this assay system.

Selection of Dose Levels for the Gene Mutation Assay
Based upon the toxicity assay, the top dose level was the 10 mM guideline limit. Four additional concentrations of the test material, the lowest of which was expected to give cell survival comparable to solvent control, were also tested in the gene mutation assay. The concentrations were spaced apart by a factor of 2. The cytotoxicity of the selected concentrations was determined concurrent with the gene mutation assay.

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 10(6) 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 10(6) 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 10(5) 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 7-9 days and the colonies were fixed/stained with methanol/crystal violet.

The mutant frequency (expressed as mutants per 10(6) clonable cells) for each replicate were calculated by the following formula (Kirkland, 1989):
MF = K x (m / c)
Where:
K = Pc x 10(6) / 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

References:
Kirkland, D. J. (editor) (1989). Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, New York, Pgs. 78-87.

O'Neill, J. P., Brimer, P. A., Machanoff, R., Hirsch, G. P., and Hsie, A. W. (1977a). A quantitative assay for mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in chinese hamster ovary cells (CHO/HGPRT): Development and definition of the system. Mutat. Res. 45, 91-101.

O'Neill, J. P. and Hsie, A. W. (1979). Phenotypic expression time of mutagen-induced 6-thioguanine resistance in chinese hamster ovary cells (CHO/HGPRT system). Mutat. Res. 59, 109-118.
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 10(6) 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 negative control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the negative control yielded significant differences.

References:
Kirkland, D. J. (editor) (1989). Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, New York, Pgs. 78-87.

Winer, B. J. (1971). Statistical Principles in Experimental Design (2nd Edition). McGraw-Hill, New York.

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Assay A1 – Preliminary Toxicity Assay
In a preliminary toxicity assay, the test material was tested at concentrations of 0 (solvent control), 2.9, 5.9, 11.7, 23.5, 46.9, 93.9, 187.8, 375.5 and 751.0 μg/ml in the absence and presence of an externally supplied metabolic activation system (S9). The highest concentration tested was based upon the 10 mM guideline limit for the assay system. The treated cultures without and with S9 activation showed little to no toxicity with the relative cell survival (RCS) values ranging from 95.7 to 116.8% in the absence of S9 and 85.5 to 108.2% in the presence of S9. Based upon the results of this assay, concentration levels of 0 (solvent control), 46.9, 93.9, 187.8, 375.5, and 751 μg/ml of the test material were selected for the initial gene mutation assay in the absence and presence of S9.

Assay B1 – Initial Mutagenicity Assay
In the initial mutagenicity assay (Assay B1), in the absence of S9, moderate to no toxicity was observed with RCS values ranging from 63.3 to 105.5% (Table 1). In the presence S9, minimal toxicity was observed with RCS values ranging from 91.3 to 109.8% (Table 2). The mutant frequencies observed in cultures treated with the test material in the absence (Table 1) and presence (Table 2) of S9 at all concentration levels were not significantly different from the concurrent solvent control values. All mutant frequencies were within a reasonable range of historical background values.

Assay C1 – Confirmatory Mutagenicity Assay
In a confirmatory assay (Assay C1), the concentrations ranged from 0 (solvent control) to 751 μg/ml in the absence and in the presence of S9. There was little to no toxicity observed, as indicated by RCS values, in the absence of S9 activation (Table 3, 87.4 to 109.8%). In the presence of S9, RCS values showed minimal to no toxicity with values ranging from 79.2 to 97.7% (Table 4). 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 solvent 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 observed concentrations of the test material in the stock dosing solutions in Assay B1 were sampled after treatment initiation and ranged from 87.9 to 98.7% of target (Appendix A) and this verified that the concentrations of nitroethane used for treatment were within acceptable values.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

pH and Osmolality

The pH and osmolality of treatment medium containing approximately 755 μg/ml of the test material and medium containing distilled water 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 = 7.66, osmolality = 292 mOsm/kg H2O; culture medium with 10% distilled water, pH = 7.59, osmolality = 290 mOsm/kg H2O).

The partitioning of the test material from the aqueous medium into the headspace was also assessed prior to treatment based on the Henrywin module (Henry’s Law Constant QSAR) of EPI Suite TM v4.1, which was developed by the EPA’s Office of Pollution

Prevention Toxics and Syracuse Research Corporation. Based on that evaluation, and the resulting Henry’s Law Constant for the test material, loss of test material to the headspace was deemed minimal under the conditions of this study. However, the caps of

the flasks used for treatment were tightly sealed during treatment to ensure all test material remained in the treatment flask during the 4 hour treatment.

Table 1 Results of the Gene Mutation Assay in CHO Cells Treated with Nitroethane in the Absence of S9 – Assay B1

 Treatment  Toxicity Assay  Mutation Assay  Cloning Efficiency (CE)  TGr Mutants/106 clonable cells
 ug/ml  RCS (%)a  Total TGr Colonies/Dishb  CE (%)c  
 Solvent control  107.8  1  79.7  0.6
 Solvent control  92.2  7  69.8  5.0
 46.9  102.3  20  56.5  17.7
 46.9  93.9  11  69.2  8.0
 93.9  103.2  18  61.5  14.6
 93.9  102.5  11  58.2  9.5
 187.8  105.5  30  54.0  27.8
 187.8  97.3  15  59.0  12.7
 375.5  71.9  9  66.0  6.8
 375.5  63.3  13  70.0  9.3
 751  93.9  8  52.8  7.6
 751  90.5  6  61.8  4.9
 Positive Controle  49.6  210  35.3  297.2d
 Positive Controle  56.5  235  39.2  300.0d

a Relative cell survival (%) = (Mean number of colonies/dish in the treated)/(Mean number of colonies/dish in the solvent control (avg. of replicates)) x 100

b TGr = 6-Thioguanine resistant

c CE (%) = (Mean number of colonies/dish)/(No. of cells seeded/dish) x 100

d The frequency of TGr mutants is significantly higher than the concurrent solvent control value (alpha=0.05) .

e 621 μg/ml EMS

Table 2 Results of the Gene Mutation Assay in CHO Cells Treated with Nitroethane in the Presence of S9 – Assay B1

 Treatment  Toxicity Assay  Mutation Assay  Cloning Efficiency (CE)  TGr Mutants x 106 Clonable Cells
  ug/ml  RCS (%)a  Total TGr Colonies/Dishb  (%)c  
 Solvent Control  104.6  13  66.8  9.7
 Solvent Control  95.4  20  70.8  15.7
 46.9  98.1  9  58.2  7.7
 46.9  96.9  20  71.5  14.0
 93.9  91.3  8  59.5  7.5
 93.9  95.1  16  61.0  13.1
 187.8  107.1  11  49.8  11.0
 187.8  104.6  29  59.8  24.2
 375.5  109.8  11  42.0  13.1
 375.5  99.9  22  61.5  17.9
 751  97.4  15  60.2  13.9
 751  100.6  12  55.5  10.8
 Positive Controlf  111.6  275  51.2  268.7e
 Positive Controlf  112.3  286  56.8  251.6e
 Positive Controlg  102.8  455  57.8  393.4e
 Positive Controlg  98.3  394  59.0  333.9e

a Relative cell survival (%) = (Mean number of colonies/dish in the treated)/(Mean number of colonies/dish in the solvent control (avg. of replicates)) x 100

b TGr = 6-Thioguanine resistant

c CE (%) = (Mean number of colonies/dish)/(No. of cells seeded/dish) x 100

e The frequency of TGr mutants is significantly higher than the concurrent solvent control value (alpha=0.05) .

f 4.0 μg/ml 20-MCA

g 8.0 μg/ml 20-MCA

Table 3 Results of the Gene Mutation Assay in CHO Cells Treated with Nitroethane in the Absence of S9 – Assay C1

 Treatment  Toxicity Assay  Mutation Assay  Cloning Efficiency (CE)  TGrMutants x 106Clonable Cells
  ug/ml  RCS (%)a  Total TGr Colonies/Dishb  (%)c  
 Solvent Control  96.5  2  87.0  1.3
 Solvent Control  103.5  4  100.3  2.5
 46.9  88.3  6  103.0  3.6
 46.9  87.4  2  85.7  1.3
 93.9  106.0  19  108.5  8.8
 93.9  109.8  20  98.5  11.3
 187.8  97.2  9  108.3  4.2
 187.8  94.4  6  119.7  2.8
 375.5  94.5  9  97.0  5.2
 375.5  97.2  4  89.2  2.8
 751  98.0  10  96.7  5.2
 751  104.6  19  90.0  11.7
 Positive Controlf  43.0  132  41.2  160.3e
 Positive Controlf  42.8  160  48.5  164.9e

aRelative cell survival (%) = (Mean number of colonies/dish in the treated)/(Mean number of colonies/dish in the solvent control (avg. of replicates)) x 100

bTGr= 6-Thioguanine resistant

cCE (%) = (Mean number of colonies/dish)/(No. of cells seeded/dish) x 100

eThe frequency of TGr mutants is significantly higher than the concurrent solvent control value (alpha=0.05) .

f621 μg/ml EMS

Table 4 Results of the Gene Mutation Assay in CHO Cells Treated with Nitroethane in the Presence of S9 – Assay C1

 Treatment  Toxicity Assay  Mutation Assay  Cloning Efficiency (CE)  TGrMutants x 106Clonable Cells
  ug/ml  RCS (%)a  Total TGrColonies/Dishb  (%)c  
 Solvent Control  108.5  13  102.0  6.4
 Solvent Control  91.5  18  116.3  7.7
 46.9  80.8  6  116.2  2.6
 46.9  79.5  16  108.5  7.4
 93.9  97.7  11  104.0  6.6
 93.9  88.1  7  108.0  3.6
 187.8  91.2  10  104.8  4.8
 187.8  93.1  4  91.0  2.2
 375.5  93.3  4  100.0  2.0
 375.5  94.3  8  96.0  4.2
 751  97.3  16  104.8  7.6
 751  79.2  10  98.2  5.1
 Positive Controlf  61.1  206  73.5  140.1e
 Positive Controlf  72.2  277  98.3  140.9e
 Positive Controlg  52.0  287  84.5  169.8e
 Positive Controlg  47.6  299  72.3  206.7e

aRelative cell survival (%) = (Mean number of colonies/dish in the treated)/(Mean number of colonies/dish in the solvent control (avg. of replicates)) x 100

bTGr= 6-Thioguanine resistant

cCE (%) = (Mean number of colonies/dish)/(No. of cells seeded/dish) x 100

eThe frequency of TGrmutants is significantly higher than the concurrent solvent control value (alpha=0.05) .

f4.0 μg/ml 20-MCA

g8.0 μg/ml 20-MCA

References:

Cifone, M. A. (1985). Relationship between increases in the mutant frequency in L5178Y TK+/- mouse lymphoma cells at low pH and metabolic activation. Environ. Mutagen. 7 (Suppl. 3), 27.

Galloway, S. M., Bean, C. L., Armstrong, M. A., Deasy, D., Kraynak, A., and Bradley, M. O. (1985). False positive In Vitro chromosome aberration tests with non mutagens at high concentrations and osmolalities. Environ. Mutagen. 7 (Suppl. 3), 48-49.

Thilagar, A. K., Kumaroo, P. V., and Kott, S. (1984). Effects of low pH caused by glacial acetic acid and hydrochloric acid on chromosomal aberrations in CHO Cells. Toxicologist 4, 51.

Applicant's summary and conclusion

Conclusions:
The test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system
Executive summary:

Nitroethane was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthineguanine-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 (solvent control) to 751 μg/ml in the absence of S9 and in the presence of S9. The highest concentration was based on the guideline limit of 10 mM for this assay system. 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. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e. distilled water). There were no statistically significant treatment-related increases in the mutant frequency in the test material-treated cultures compared to the solvent control cultures in either the absence or presence of S9. The results of this CHO/HGPRT forward gene mutation assay with nitroethane 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.