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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a GLP bacterial reverse mutation study conducted according to OECD TG 471, it was concluded that n-propyl propionate did not induce reverse gene mutation in the selected bacterial tester strains in the presence and absence of S9 mircosomal fraction obtained from rat liver homogenate.

In a GLP in vitro chromosomal aberration assay conducted according to OECD TG 476 and EPA OPPTS 870.5375 utilizing rat lymphocytes, n-propyl propionate was considered to be negative.

In a third GLP study, conducted according to OECD TG 476 (In vitro Mammalian Cell Gene Mutation Test) with category member n-pentyl propionate, there was no evidence of genotoxicity at the highest, non-cytotoxic, concentrations in the presence or absence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 471, EPA OPPTS 870.5100, EU Method and in accordance with the Principles of Good Laboratory Practice (GLP).
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Some minor deviations were noted, however these observations did not have an adverse impact on the quality and integrity of the study
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
same as above
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Salmonella typhimurium tester strains in use at Covance were received directly from Dr. Bruce Arnes, Department of Biochemistry, University of California, Berkeley. Escherichia coli tester strain, WP2uvrA, was received from The National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).
- Properly maintained: 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:
S9
Test concentrations with justification for top dose:
Concentrations tested in the mutagenicity assay were selected based on the results of the rangefinding study. The concentrations tested with all tester strains were 33.3, 100,333, 1000, 2500 and 5000 µg per plate in the presence of S9 mix and 10.0, 33.3, 100,333, 1000,2500 and 5000 µg per plate in the absence of S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO - Vehicle controls were plated for all tester strains in the presence and absence of S9 mix. The vehicle control was plated, using a 50 µL aliquot of dimethylsulfoxide (DMSO) (equal to the maximum aliquot of test article plated), along with a 100 µL aliquot of the appropriate tester strain and a 500 µL aliquot of S9 mix (or 0.1 M phosphate buffer, when necessary), on selective agar
- Justification for choice of solvent/vehicle: recommended vehicle by various regulatory agencies
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: benzo[a]pyrene, 2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR-191, 4-nitroquinoline-N-oxide. All positive control articles were manufactured by Sigma Chemical Company, exception of 2-aminosanthracene and 2-nitrofluorene (Aldrich Chemical Company)
Details on test system and experimental conditions:
Frozen permanent stocks were prepared by growing fresh overnight cultures, adding DMSO (0.09 ml/ml of culture) and freezing away appropriately vialed aliquots. Frozen permanent stocks of tester strains were stored at -60°C to -80°C. Master plates of tester strains were prepared by streaking each tester strain from a frozen permanent stock onto minimal agar appropriately supplemented with either histidine and biotin or tryptophan, and for strains containing the pKM101 plasmid, ampicillin. Tester strain master plates were stored at >0°C to 10°C.

Overnight cultures for use in all testing procedures were inoculated by transferring a colony from the appropriate master plate to a flask containing culture medium. Inoculated flasks were placed in a shaker/incubator programmed to begin operation (shaking, 125 ± 25 rpm; incubation, 37 ± 1 °C) so that overnight cultures were in log phase or late log phase when turbidity monitoring began.

To ensure that cultures were harvested in late log phase, the length of incubation was determined by spectrophotometric monitoring of culture density. Cultures were harvested once a predetermined density was reached which ensured that cultures had reached a density of at least 1.0 x 10(9) cells per ml and had not overgrown. Cultures were removed from incubation when target density was reached and held at >0°C to 10°C until used in the assay.

The broth used to grow overnight cultures of the tester strains was Vogel-Bonner salt solution supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2 (dry powder). Bottom agar (25 ml per 15 x 100 mm petri dish) was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. Top (overlay) agar was prepared with 0.7% (w/v) agar and 0.5% (w/v) NaCl and was supplemented with 10 ml of 0.5 mM histidine/biotin solution per 100 ml agar for selection of histidine revertants or 0.5 mM tryptophan solution per 100 ml of agar for selection of tryptophan revertants. For an agar overlay, 2.0 ml of supplemented top agar was used.

The range finding study was performed using tester strains TAl 00 and WP2uvrA in both the presence and absence of S9 mix. Ten concentrations of test article were tested at one plate per concentration. The test article was checked for cytotoxicity up to a maximum concentration of 5000 µg per per plate.

The mutagenicity assay was performed using tester strains TA98, TAl 00, TA1535, TA1537, and WP2uvrA both in the presence and absence of S9 mix along with appropriate vehicle and positive controls. In the preincubation methodology, S9 mix (or phosphate buffer, where appropriate), tester strain, and test article were preincubated prior to addition of molten agar. The agar and preincubation reaction mixture were mixed and then overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. Test article, vehicle controls, and positive controls were plated in triplicate.

These procedures were used in both the range finding study and the mutagenicity assay. Each plate was labeled with a code that identified the test article, test phase, tester strain, activation condition and concentration. S9 mix was prepared immediately prior to use.

When S9 mix was required, 500 µl of S9 mix was added to 13 x 100 mm glass culture tubes, which had been pre-heated to 37 ± 1°C. To these tubes were added 100 µl of tester strain and 50 µl of vehicle or test article. When S9 mix was not required, 500 µl of 0.1 M phosphate buffer was substituted for S9 mix. After required components had been added, the mixture was vortexed and allowed to incubate for 20 ± 2 minutes at 37 ± 1°C. Two ml of molten selective top agar was then added to each tube, and the mixture was vortexed and overlaid onto the surface of 25 ml of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay solidified, plates were inverted and incubated for 52 ± 4 hours at 37 ± 1°C. Positive control articles were plated using a 50 µl plating aliquot.
Evaluation criteria:
Plates not evaluated immediately following the incubation period were held at >0°C to 10 °C until colony counting and bacterial background lawn evaluation could take place.

Condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that concentration. Lawns were scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C)
also were noted.

Revertant colonies were counted either by automated colony counter or by hand.
Statistics:
Descriptive statistical methods were used
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
In the range finding test, ten concentrations of test article, from 6.67 to 5000 pg per plate, were tested. Reduced or absent bacterial background lawns were observed at 5000 pg per plate in the presence of S9 mix and at 3330 µg per plate and above in the absence of S9 mix. No concentration-related decreases
in the number of revertants per plate were observed with WP2uvrA in the presence or absence of S9 mix. Decreases in the number of revertants were observed with TA1 00 at 5000 µg per plate in the presence of S9 mix and at 3330 µg per plate and above in the absence of S9 mix.

The tester strains used in the preincubation mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted with six concentrations of test article in the presence of S9 mix and seven concentrations in the absence of S9 mix along with concurrent vehicle and positive controls using three plates per concentration.

Concentrations tested in the mutagenicity assay were selected based on the results of the rangefinding study. The concentrations tested with all tester strains were 33.3, 100,333, 1000, 2500 and 5000 µg per plate in the presence of S9 mix and 10.0,33.3, 100,333, 1000,2500 and 5000 µg per plate in the absence of S9 mix.

In the initial mutagenicity assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.

In the confirmatory mutagenicity assay, all data generated with tester strain WP2uvrA were acceptable and no positive increases in the mean number of revertants per plate were observed with WP2uvrA in either the presence or absence of S9 mix. In this trial, no valid data were generated with any of the Salmonella tester strains in either the presence or absence of S9 mix due to multiple technical problems. In another confirmatory mutagenicity assay, enhanced bacterial lawn growth was observed with TA100 in the presence and absence of S9 mix. In addition, the mean vehicle control values were not within the acceptable ranges for tester strain TA98 in the presence and absence of S9 mix, and for TA1535 and TA1537 in the absence of S9 mix. The mean vehicle control values for tester strains TA1535 and TA1537 in the presence of S9 mix were acceptable, however the results for these strains could not be considered valid since the mean vehicle control values without S9 indicated that the strains were not functioning properly. For these reasons, any data generated with the Salmonella strains in Trial 26100-C1 were not used to evaluate the test article. The test article was re-tested with the Salmonella strains in Trial 261 00-D1. Also, in order to clarify a discrepancy in toxicity observed at the maximum concentration tested in Trials 26 1 00-B 1 and 26 1 00-C 1, the test article was re-tested with tester strain WP2uvrA in the absence of S9 mix in Trial 26100-D1.

In the repeat mutagenicity assay (Trial 26100-D1), all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. All criteria for a valid study were met.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Interpretation of results (migrated information):
negative

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay indicate that under the conditions of this study, the test article, N-Propyl Propionate, did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9).
Executive summary:

The objective of this study was to evaluate the test article N-Propyl Propionate, for the ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains ofSalmonella typhumuriumand at 2) the tryptophan locus ofEscherichia colistrain WP2uvrA. This assay satisfied the following guidelines: U.S EPA (1998), EEC (2000), and OECD (1997).

 

The tester strains used in the mutagenicity assay wereSalmonella typhumuriumtester strains TA98, TA100, TA1535, and TA1537 andEscherichia colitester strain WP2uvrA. The assay was conducted with a minimum of six concentrations of test article in the presence of S9 mix and absence of S9 mix, along with concurrent vehicle and positive controls using three plates per concentration. The concentrations tested in the mutagenicity assay were 33.3, 100, 333, 1000, 2500 and 5000 μg per plate in the presence of S9 mix and 10.0, 33.3, 100, 333, 1000, 2500 and 5000 μg per plate in the absence of S9 mix.

 

The results of the initial mutagenicity assay were confirmed in an independent experiment. The results of theSalmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay indicate that under the conditions of this study, the test article, N-Propyl Propionate, did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 473, EPA OPPTS 870.5375, EU Method B.10 and in accordance with the Principles of Good Laboratory Practice (GLP).
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: rat
Details on mammalian cell type (if applicable):
Blood samples were collected by cardiac puncture, following euthanasia with carbon dioxide. In the assay, blood samples from individual rats were pooled and whole blood cultures were set up in RPMI 1640 medium (with 25 mM HEPES, GIBCO, Grand Island, New York) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO), antibiotics and antimycotics (Fungizone 0.25 μg/ml; penicillin G, 100 u/ml; and streptomycin sulfate, 0.1 mg/ml; GIBCO), 30 μg/ml PHA (HA16, Murex Diagnostics Ltd., Dartford, England), and an additional 2 mM L- glutamine (GIBCO). Cultures were initiated by inoculating approximately 0.5 ml of whole blood/5 ml of culture medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Assay A1 - In this assay, cultures were treated with the test material in the absence and presence of S9 activation for 4 hours at concentrations of 0 (solvent control), 18.75, 37.5, 75, 150, 300, 600, and 1200 μg/ml. Based up on these results, cultures treated for 4 hours with targeted concentrations of 0 (solvent control), 300, 600, and 1200 mg/ml in the absence and presence of S9 activation and cultures treated for 24 hours with 0 (solvent control), 75, 150, and 300 mg/ml were selected for determining the incidence of chromosomal aberrations.
The selected concentrations of the test material in the treatment solutions were diluted in an appropriate solvent and analyzed by gas chromatography (GC) with flame ionization detection (FID).
Vehicle / solvent:
The solvent selected for dissolving the test material (i.e., dimethyl sulfoxide) was used as the negative control treatment. The test material was first dissolved in dimethyl sulfoxide (DMSO, Sigma) and further diluted (1 :100) with the treatment media to obtain the desired concentrations.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Remarks:
Mitomycin C (MMC, Sigma, St. Louis, Missouri, CAS No. 50-07-7) was used as the pos itive control chemical for the nonactivation assay at a concentration of 0.5 μg/ml (4 hour treatment) or 0.05 and 0.075 μg/ml (24 hour treatment).
Positive control substance:
other: Mitomycin C and cyclophosphamide
Remarks:
Cyclophosphamide monohydrate (CP, Sigma, CAS No. 6055-19-2) was the positive control for the activation assay at final concentrations of 4 and 6 μg/ml.
Details on test system and experimental conditions:
Treatment Procedure without Metabolic Activation:
Short Treatment -
Forty-eight hours (± 1 hour) after initiation of the cultures, the cell suspension was dispensed into 15 ml sterile disposable centrifuge tubes (5.5 ml/tube, two cultures per dose level). The cells were sedimented by centrifugation and the culture medium removed and saved. The cells were exposed to medium (RPMI 1640, HEPES, and antibiotics) containing the test or positive or solvent control treatments for 4 hours (± 30 minutes) at 37°C and the exposures were terminated by washing the cells with culture medium. The cells were then placed in individual sterile disposable tissue culture flasks (T-25) along with approximately 4.5 ml of the original culture medium until the time of harvest. The cultures were harvested 24 hours (± 1 hour) after treatment initiation (i.e., 20 hours, ± 1 hour after treatment termination).

Continuous Treatment -
Cultures were treated continuously with the test material for 1.5X normal cell cycle length. The solvent control, positive control, and test material were added directly to the culture flasks 48 hours (± 1 hour) after initiation of the cultures and the cultures were harvested 24 hours (± 1 hour) later.

Treatment Procedure using Metabolic Activation (S9):
Only the short treatment (4 hours ± 30 minutes) procedure was used with S9. Forty eight hours (± 1 hour) after initiation of the cultures, the cell suspension was dispensed into 15 ml sterile disposable centrifuge tubes (5.5 ml/tube, two cultures per dose level). The cells were sedimented by centrifugation and the culture medium removed and saved. The cells were exposed to medium (RPMI 1640, HEPES,
antibiotics, and the S9 mix) containing the test or positive or solvent control treatments for 4 hours (± 30 minutes) at 37°C and the exposure were terminated by washing the cells with culture medium. The cells were then placed in individual sterile disposable tissue culture flasks (T-25) along with 4.5 ml of the original culture medium until the time of harvest. The cultures were harvested at 24 hours (± 1 hour) after treatment initiation (i.e., 20 hours, ± 1 hour after treatment termination).

Colcemid (1 μg/culture) was added 2-3 hours prior to harvest. The cells were swollen by hypotonic treatment (0.075 M KCl), fixed with methanol:acetic acid (3:1), dropped on microscope slides, and stained in Giemsa.
Evaluation criteria:
For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the solvent controls. The aberration frequency in the solvent control should be within reasonable limits of the laboratory historical values. A test chemical is considered positive in this assay if it induces a significant dose-related and reproducible increase in the frequency of cells with aberrations.
Statistics:
Standard statistical methods were employed
Species / strain:
lymphocytes: rat
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In this assay, cultures were treated with the test material in the absence and presence of S9 activation for 4 hours at concentrations of 0 (solvent control), 18.75, 37.5, 75, 150, 300, 600, and 1200 μg/ml. Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration of 9.4 µg/ml. The highest concentration evaluated was based on the limit dose of 10 mM in this assay system. The analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 87.9 to 98.3% of the target.

Without and with metabolic activation (4 hour treatment), the cultures did not display any toxicity as measured by mitotic indices. In the absence of S9, the mitotic indices for the treated cultures ranged from 95.0 to 123.0% relative to the solvent control values. In the presence of S9, the mitotic indices of the treated cultures ranged from 84.7 to 114.7% as compared to the solvent control values. Cultures treated continuously for 24 hours in the absence of S9 activation showed moderate to no signs of toxicity in the treated cultures as measured by mitotic indices. Based up on these results, cultures treated for 4 hours with targeted concentrations of 0 (solvent control), 300, 600, and 1200 mg/ml in the absence and presence of S9 activation and cultures treated for 24 hours with 0 (solvent control), 75, 150, and 300 µg/ml were selected for determining the incidence of chromosomal aberrations.

Among the cultures treated with the positive control chemicals for 4 hours, 0.5 μg/ml of Mitomycin C and 4 μg/ml of Cyclophosphamide were selected for evaluation of aberrations in the absence and presence of S9, respectively. Cultures treated with 0.05 µg/ml Mitomycin C were selected for evaluation to serve as the positive control for the 24 hour assay in the absence of S9.

There were no significant increases in the incidence of polyploidy cells in any of the test material treated cultures as compared to the solvent control values.

In the 4 hour non-activation assay, the frequency of cells with aberrations in the solvent control was 1.0% and the corresponding values at treatment levels of 300, 600, and 1200 μg/ml were 0.0, 1.0, and 0.5%, respectively. In the activation assay, cultures treated with the test material at concentrations of 300, 600, and 1200 μg/ml had aberrant cell frequencies of 1.0, 2.0, and 1.0%, respectively as compared to the solvent control value of 0.0%. Statistical analyses of these data did not identify significant differences between the solvent control and any of the treated cultures without or with S9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the laboratory historical background range.

In the non-activation assay, where cultures were treated continuously for 24 hours with the test material, the frequencies of aberrant cells in the solvent control was 1.0% and the corresponding values at concentration levels of 75, 150, and 300 µg/ml were 0.0, 1.0, and 1.5%, respectively. There were no statistically significant differences between the test material treated cultures and the solvent control values and all values were within the laboratory historical background range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in Mitomycin C (- S9, 4 hour treatment), Cyclophosphamide (+ S9) and Mitomycin C (- S9, 24 hour treatment) cultures were 45.0%, 36.0%, and 13.7%, respectively.

A second assay with treatment of cultures in the presence of S9 was not considered necessary since the results of the initial test yielded clearly negative results.
Remarks on result:
other: other:
Remarks:
Migrated from field 'Test system'.

The pH of treatment medium containing approximately 1220 μg n-propyl propionate/ml of the test material (10 mM limit of assay system) and medium containing 1% solvent (DMSO) was determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE Aä freezing point osmometer, respectively. 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 = 7.15, osmolality = 440 mOsm/kg H2O; culture medium with 1% DMSO, pH = 7.08, osmolality = 409 mOsm/kg H2O).

Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that under the experimental conditions used, n-propyl propionate was non-genotoxic in this in vitro chromosomal aberration test.
Executive summary:

n-Propyl Propionate (propionic acid, propyl ester) was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (solvent control) to 1200 µg n-propyl propionate per ml of culture medium. The duration of treatment was 4 and 24 hours without S9 and 4 hours with S9. The highest concentration was based on the limit dose of 10 mM in this assay system. Based upon the mitotic indices, cultures treated for 4 hours with targeted concentrations of 0 (solvent control), 300, 600, and 1200 µg/ml in the absence and presence of S9 activation and cultures treated for 24 hours with 0 (solvent control), 75, 150, and 300 µg/ml were selected for determining the incidence of chromosomal aberrations. There were no significant increases in the frequencies of cells with aberrations in either the presence or absence of S9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of abnormal cells in all assays. Based upon these results, n-propyl propionate was considered to be non-genotoxic in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2012
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 accordance with the Principles of Good Laboratory Practice (GLP).
Justification for type of information:
See category document in section 13
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
Concentration analyses was not performed for the positive control substances (ethyl methanesulfonate and 3-methylcholantherene) used in this study
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
The in vitro mammalian cell gene mutation test 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 human lymphoblastoid cells. In these cell lines the most commonly-used genetic endpoints 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 X-chromosomes.
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 supplemented with sodium bicarbonate, antibiotics, and L-glutamine was the basic medium. Basic medium supplemented with 10% fetal bovine serum (FBS) was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells.
- 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 1254 induced rat liver S-9 homogenate
Test concentrations with justification for top dose:
Preliminary cytotoxicity test - 6, 11, 23, 45, 90, 180, 361, 721 and 1442 µg/ml
Initial gene mutation assay - 14, 46, 144, 456 and 1442 µg/ml (presence of metabolic activation)
Initial gene mutation assay - 14, 46, 100, 200, 300, 400 and 446 µg/ml (absence of metabolic activation)
Confirmatory gene mutation assay - 18, 53, 160 and 1442 µg/ml (presence of metabolic activation)
Confirmatory gene mutation assay - 18, 53, 160, 300, 350 and 400 µg/ml (absence of metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: recommended vehicle by various regulatory agencies
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Remarks:
Ethyl methanesufonate (EMS), Batch No. BCBF0736V and 3-Methylcholantherene (3-MCA), Batch No. MKBB1176. (Both sourced from Sigma Aldrich Co., St. Louis, MO 63103, USA)
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Remarks:
3-Methylcholantherene (3-MCA) at 8 µg/ml and Ethyl methanesulfonate (EMS) at 600 µg/ml (Density 1.17 g/ml)
Details on test system and experimental conditions:
Chinese hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275, American Type Culture Collection, USA) with a model chromosome number 20 and a population doubling time of 10 to 14 hours was used. Batch No. 3 of this CHO-K1 cell line was tested for the absence of mycoplasma contamination at Mycoplasma Laboratory, Statens Serum Institut, Denmark and certified free of mycoplasma contamination on 21 July 2011. Cells were grown in tissue culture flasks at 37 ± 1°C in a carbon dioxide incubator (5 ± 0.2 % carbon dioxide in air). Stock cultures of the CHO-K1 cell line were stored at the laboratory as frozen permanents in liquid nitrogen.
Ham’s F-12 medium supplemented with sodium bicarbonate, antibiotics, and L-glutamine was the basic medium. Basic medium supplemented with 10% fetal bovine serum (FBS) was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells. Basic medium without serum was the treatment medium and was used for target cell exposure to the test substance and controls. Cloning medium was basic medium supplemented with 20 % FBS and was used for the determination of cell viability or plating/cloning efficiency. Selective medium was basic medium supplemented with 20 % FBS and the selective agent 6-thioguanine (6-TG) at 35 µM and was used for the selection of mutants.
Aroclor 1254 induced rat liver S-9 homogenate was used as the metabolic activation system. The S-9 homogenate was prepared from male Wistar rats induced with a single intraperitoneal injection of Aroclor 1254, 5 days prior to sacrifice. The S-9 homogenate was prepared in batches and stored in a deep freezer maintained at -68 to -86 °C. Each batch of S-9 homogenate was characterized for its ability to metabolize the promutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain, protein content using modified Lowry Assay, and sterility. To check the sterility, the liver homogenate was streaked onto nutrient agar plates, in duplicate, and incubated for 48 hours at 37 ± 1 °C. S-9 homogenate was thawed immediately before use and mixed with the co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in PBS.
The co-factor solution was prepared by dissolving the following in 9 ml PBS for the cytotoxicity test and 27 ml PBS for the mutation assays. The co-factor solution was then sterilized using a 0.2 µm disposable syringe filter. The S-9 mix was prepared by mixing 1 ml and 3 ml S-9 homogenate with 9 ml and 27 ml of the co-factor solution, for the cytotoxicity test and the mutation assays, respectively. It was kept in an ice bath and used within one hour.
Stability of the dose formulations of the test substance at 100 and 150000 µg/ml was established in DMSO after 4 hours using a validated analytical method.
Preliminary Cytotoxicity Test –
Two sets of 25 cm2 tissue culture flasks each set having 10 flasks were prepared to represent 4-hour exposure in the presence (set 1) and absence (set 2) of metabolic activation. Exponentially growing CHO-K1 cells were plated approximately at 10(6) cells/flask with 5 ml complete medium and incubated for 24 hours. A stock dosing solution of 144200 µg/ml of the test substance prepared in DMSO for the solubility test was further diluted in DMSO immediately before use to get 600, 1100, 2300, 4500, 9000, 18000, 36100, and 72100 µg test concentrations per ml of DMSO. Target cells were exposed to the following 9 concentrations, in the presence and absence of metabolic activation, up to a maximum of 1442 µg/ml which is equivalent to the 10 mM test substance concentration along with the DMSO control, 6, 11, 23, 45, 90, 180, 361, 721 and 14422 µg/ml.
For the initial pH and osmolality determination, 50 µl of the stock / required dilution of the test substance and the vehicle control were transferred in to pre-labeled tubes containing 4.5 ml (presence of metabolic activation) and 5 ml (absence of metabolic activation) of treatment medium. For the test in the presence of metabolic activation, 0.5 ml of S-9 mix was added to each tube to achieve a concentration of 10 % (v/v) of S-9 mix. The tube contents were mixed and the pH and osmolality of the test solutions was determined.
For cytotoxicity assessment, medium from the target cell flasks was removed by aspiration and replaced with 4.5 ml and 5 ml of treatment medium for the first and second set of flasks, respectively. For the test in the presence of metabolic activation, 0.5 ml of S-9 mix was added to the respective flasks to achieve a final concentration of 10 % (v/v) in the medium.
Fifty (50 µL) of the vehicle or the stock / dilutions of the test substance were mixed with the medium in the respective flasks to get the required test concentrations per ml of the medium as well as the vehicle control. The flasks were incubated for 4 hours to expose the cells to treatment. After the treatment period, the flasks were observed for any precipitation, the exposed test solution was transferred to pre-labeled tubes and the pH and osmolality was determined.
Mutation assay – One hundred fifty micro liters (150 µl) of DMSO was used per 15 ml of treatment medium as the vehicle control in each of the experiments and designated as Group 1 (G1). DMSO was used to prepare the stocks of the following activation specific positive controls in each of the experiments and designated as Group 7 (G7), in the presence of Exogenous Metabolic Activation (+S-9): 3-Methylcholantherene (3-MCA) at 8 µg/ml and in absence of Exogenous Metabolic Activation (-S-9): Ethyl methanesulfonate (EMS) at 600 µg/ml (Density 1.17 g/ml).
Initial Gene Mutation Assay - Based on the results of the cytotoxicity test, the following test concentrations were selected for testing in the initial gene mutation assay in the presence of metabolic activation: 14, 46, 144, 456 and 1442 µg/ml and 14, 46, 100, 200, 300, 350, 400 and 446 µg/ml in the absence of metabolic activation.
Confirmatory Gene Mutation Assay - Since the results of the initial gene mutation assay were negative, the following test concentrations were selected for testing in the confirmatory gene mutation assay in the presence of metabolic activation: 18, 53, 160, 228, 481 and 1442 µg/ml and 18, 53, 160, 300, 350 and 400 µg/ml in the absence of metabolic activation.
The test flasks were labeled to indicate the details of the study and the assay phase. Exponentially growing CHO-KI cells were plated in duplicate in 15 ml of complete medium at a density of approximately 3x10(6) cells / 75 cm2 flask and incubated for 24 hours.
All test substance and positive control concentrations were prepared immediately before use in sterile test tubes. The target cells in duplicate cultures were exposed to the vehicle, positive controls and selected concentrations of the test substance for 4 hours in the presence and absence of metabolic activation. Test substance concentrations were prepared at room temperature and under yellow light. The medium from each target cell flask was removed by aspiration and replaced with 13.5 ml and 15 ml of treatment medium for the experiment in the presence and absence of metabolic activation, respectively. For the experiment incorporating metabolic activation, 1.5 ml of S-9 mix was added to give a final concentration of 10 % (v/v) in the test solutions. One hundred fifty micro liters (150 µl) each of the vehicle control, respective positive controls, or stock/dilution of the test substance were transferred to respective flasks and gently mixed and the flasks were kept for incubation.
Assessment of Parallel Cytotoxicity and Expression of the Mutant Phenotype - After the treatment period, replicate cultures from controls and each treatment level were trypsinized, detached with 5 ml complete medium, and counted using a hemocytometer. About 200 cells from each replicate of the controls and each treatment level were plated into T-25 cm2 flasks in triplicate with cloning medium to determine ACE and to express parallel cytotoxicity based on RCE. After 9 days of incubation, the colonies were stained with methylene blue and counted for cloning efficiency. For expression of the mutant phenotype, the cells from the replicate cultures were sub-cultured in complete medium in duplicate, at a density of approximately 10(6) cells/25 or 75 cm2 flasks and incubated. The cells were subcultured as above at a 2-3 day interval and carried out for the 9 day expression period. After this time, the mutant phenotype was selected.
Selection of the Mutant Phenotype and Plating for Cloning Efficiency - Replicate cultures from contrkols and each treatment level were trypsinized, detached with 5 mL complete medium, pooled, and counted using a hemocytometer. For selection of the 6-Thioguanine (6TG) resistant phenotype, cells from each of the replicate cultures were plated in to 10 flasks at a density of approximately 2 x 10(5) cells/25 cm2 flask (total of 2 x 10(6) cells) in selective medium and incubated for 10 and 7 days for the initial and confirmatory gene mutation assays, respectively. For cloning efficiency determination at the time of selection, cells from each of the replicate cultures were plated approximately at 200 cells/25 cm2 flask in triplicate in cloning medium and incubated for 10 and 7 days for the initial and confirmatory gene mutation assays, respectively.
Staining – The colonies were stained with 0.5% methylene blue and counted for both cloning efficiency and mutant selection after 10 days of incubation.
Evaluation criteria:
Assessment of cytotoxicity –
Medium from each flask was removed by aspiration and the cell monolayer was rinsed with PBS, trypsinized. and then the cells were suspended in 5 ml complete medium and counted using a hemocytometer. About 200 cells from the control and each treatment level were plated into T-25 cm2 flasks with 5 ml cloning medium in triplicate and incubated for 10 days. After incubation, medium from each flask was aspirated and the cells were stained with methylene blue and the colonies counted manually to determine absolute cloning efficiency (ACE) and cytotoxicity was expressed relative to the vehicle treated control (relative cloning efficiency – RCE).
There are several criteria for determining a positive result, such as a concentration related, or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. A test substance, for which the results do not meet the above criteria is considered non mutagenic in this system
Statistics:
Standard statistical methods were employed
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:
The S-9 homogenate was found to be sterile and active as evidenced by its ability to metabolize the promutagens, 2-aminoanthacene and benzo(a)pyrene to mutagens using S. typhimurium strain TA 100 and the protein content of the S-9 homogenate was 26.6 mg/ml. The test substance was stable in DMSO at 100 and 150000 µg/ml after 4 hours at room temperature. The results of the concentration analysis for the initial gene mutation assay indicate that the actual mean concentrations of the analyzed dose levels were between 98.4 and 107.2 % of their respective nominal target concentrations confirming that the concentration of the test substance was within acceptable limits (85 to 115 % of nominal concentrations). The results of the concentration analysis for the confirmatory gene mutation assay indicate that the actual mean concentrations of the analyzed dose levels were between 98.5 and 110 % of their respective nominal target concentrations, confirming that the concentration of the test substance was within acceptable limits (85 to 115 % of nominal concentrations).
Cytotoxicity Test - No precipitation in the test solutions was observed at any of the test concentrations either in the presence or in the absence of metabolic activation. At the end of 4-hour exposure period, the pH of the test solutions at the highest tested concentration of 1442 µg/ml was 6.9 with 7.02 in the presence and absence of metabolic activation, respectively while the corresponding pH values in the DMSO control were 6.96 and 7.1 in the presence and absence of metabolic activation, respectively. At the end of 4-hour exposure period, the osmolality of the test solution at the highest tested concentration of 1442 µg/ml was 0.450 and 0.448 OSMOL/kg in the presence and absence of metabolic activation, while in the DMSO control, the corresponding osmolality was 0.474 and 0.456 OSMOL/kg in the presence and absence of metabolic activation. At the end of 4-hour exposure period, the RCE values in the presence of metabolic activation, ranged from 26 to 91 % while in the absence of metabolic activation, RCE values ranged from 10 to 92 % compared to the vehicle control. At 721 and 1442 µg/ml test concentrations, there was no cell growth in the absence of metabolic activation.
Initial Mutation Assay - There was no evidence of excessive cytotoxicity (i.e., <10 % RCE) at any of the tested concentrations either in the presence or absence of metabolic activation. The RCE values in the presence of metabolic activation, ranged from 21.8 to 85.9 % while in the absence of metabolic activation, ranged from 11.3 to 86.7 % compared to the vehicle control. The frequency of mutants in the DMSO control was within the range of the in-house historical control data. The test substance did not cause a significant increase in the frequency of mutants compared to the vehicle control in the presence or absence of metabolic activation at any of the tested concentrations. Under similar conditions the positive controls 3-methylcholantherene (3-MCA) and ethyl methanesulfonate (EMS) both induced significant increases in the mutant frequency as compared with the vehicle control.
Confirmatory Mutation Assay - There was no evidence of excessive cytotoxicity (<10% RCE) at any of the tested concentrations either in the presence or absence of metabolic activation. The RCE values in the presence of metabolic activation, ranged from 22.6 to 92.4 % while in the absence of metabolic activation, ranged from 14.5 to 83.4 % compared to the vehicle control. The frequency of mutants in the DMSO control was within the range of the in-house historical control data. The test substance did not cause a significant increase in the frequency of mutants compared to the vehicle control in the presence or absence of metabolic activation at any of the tested concentrations. Under similar conditions the positive control 3-methylcholantherene (3-MCA) and ethyl methanesulfonate (EMS) both induced a significant increase in the mutant frequency as compared with the vehicle control.
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Interpretation of results (migrated information):
negative

The results of the forward gene mutation assay at the hprt locus with n-pentyl propionate indicate that under the conditions of this study, the test substance was non-mutagenic when evaluated in the presence or absence of an externally supplied metabolic activation (S9) system.
Executive summary:

The genotoxic potential of the test substance n-pentyl propionate to induce gene mutation in mammalian cells was evaluated using Chinese hamster ovary (CHO) cells. The study consisted of a preliminary toxicity test, an initial gene mutation assay, and a confirmatory gene mutation assay. Each of these mutation assays comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver). N-pentyl propionate formed a solution in dimethyl sulfoxide (DMSO) at 150000 µg/ml and was found to be stable in DMSO after 4 hours at the concentrations of 100 and 150000 µg/ml. In a preliminary cytotoxicity test n-pentyl propionate did not cause a significant cell growth inhibition as evaluated by relative cloning efficiency (RCE) up to the highest tested concentration of 1442 µg/ml (equivalent to 10 mM) in the presence as well as in the absence of metabolic activation. However, in the absence of metabolic activation, there was significant cell growth inhibition as RCE at 361 µg/ml and at 721 and 1442 µg/ml, there was no cell growth, due to excessive toxicity of the test substance. The test substance did not precipitate in the treatment medium up to 1442 µg/ml, and did not cause any appreciable change in the pH or osmolality of the test solutions at the end of the 4-hour exposure to treatment either in the presence or in the absence of metabolic activation.

In the initial gene mutation assay, CHO cells were exposed to the test substance in duplicate at concentrations of 14, 46, 144, 456, and 1442 µg/ml of the medium for 4 hours in the presence and at 14, 46, 100, 200, 300, 350, 400 and 456 µg/ml in the absence of metabolic activation. In the confirmatory gene mutation assay, CHO cells were exposed to the test substance in duplicate at concentrations of 18, 53, 160, 481, and 1442 µg/ml of the medium for 4 hours in the presence and at 18, 53, 160, 300, 350 and 400 µg/ml in the absence of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and appropriate positive controls i.e., 3-methylcholantherene in the presence of metabolic activation and ethyl methanesulphonatemethanesulfonate in the absence of metabolic activation were also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test material treated cultures either in the presence or absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequency of mutants, under identical conditions and the concurrent vehicle control cultures values were within laboratory historical controls.

The results of concentration analysis of dose formulations from the initial as well as confirmatory gene mutation assays confirmed that the concentration of the test substance was within acceptable limits.

 

The results of the forward gene mutation assay at the hprt locus with n-pentyl propionate indicate that under the conditions of this study, the test substance was non-mutagenic when evaluated in the presence or absence of an externally supplied metabolic activation (S9) system

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The tester strains used in the mutagenicity assay wereSalmonella typhumuriumtester strains TA98, TA100, TA1535, and TA1537 andEscherichia colitester strain WP2uvrA. The assay was conducted with a minimum of six concentrations of test article in the presence of S9 mix and absence of S9 mix, along with concurrent vehicle and positive controls using three plates per concentration. The concentrations tested in the mutagenicity assay were 33.3, 100, 333, 1000, 2500 and 5000 μg per plate in the presence of S9 mix and 10.0, 33.3, 100, 333, 1000, 2500 and 5000 μg per plate in the absence of S9 mix.

 

The results of theSalmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay indicate that under the conditions of this study, the test article, N-Propyl Propionate, did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9).

N-Propyl Propionate was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (solvent control) to 1200µg n-propyl propionate per ml of culture medium. The duration of treatment was 4 and 24 hours without S9 and 4 hours with S9. The highest concentration was based on the limit dose of 10 mM in this assay system. Based upon the mitotic indices, cultures treated for 4 hours with targeted concentrations of 0 (solvent control), 300, 600, and 1200µg/ml in the absence and presence of S9 activation and cultures treated for 24 hours with 0 (solvent control), 75, 150, and 300µg/ml were selected for determining the incidence of chromosomal aberrations. There were no significant increases in the frequencies of cells with aberrations in either the presence or absence of S9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of abnormal cells in all assays. Based upon these results, n-propyl propionate was considered to be non-genotoxic in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

The genotoxic potential of category member n-pentyl propionate to induce gene mutation in mammalian cells was evaluated using Chinese hamster ovary (CHO) cells. There was no evidence of induction of gene mutations in any of the treated cultures either in the presence or absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequency of mutants, under identical conditions and the concurrent vehicle control cultures values were within laboratory historical controls. Under the conditions of this study, the catgory member n-pentyl propionate was non-mutagenic when evaluated in the presence or absence of an externally supplied metabolic activation (S9) system.


Justification for classification or non-classification

As negative results were noted in the Bacterial Reverse Mutation test (AMES) and rat lymphocyte chromosome aberration test with n-propyl propioate, and in the mammalian cell mutagenicity assay with category member n-pentyl propionate, and as per the Guidance to Regulation (EC) No. 1272/2008 on Classification, Labelling and Packaging of substances and mixtures, n-butyl propionate will not be classified for genotoxicity.