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Genetic toxicity in vitro

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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:
2009-2010
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 B 13/14 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 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
The purpose of this study was to evaluate the mutagenic potential of UCAR n-Pentyl Propionate and/or its metabolites by utilizing a pre-incubation method to measure its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA pKM101 in the presence and absence of S9 activation.
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):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 metabolic activation
Test concentrations with justification for top dose:
Initial Toxicity Mutation assay - 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Confirmatory Mutation assay - 100, 266, 707, 1880 and 5000 µg/plate.
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:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Nitrofluorene, 2-Aminoanthracene, 9-Aminoacridine, 4-Nitroquinoline-1-oxide and sodium azide
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

The petri-dishes were labeled to indicate the study number, strain number, treatment group, trial number, plate number and activation. For the initial toxicity mutation test two replicates were used while for the confirmatory mutation test three replicates were used. Mutation assay was performed following the pre-incubation procedure. The following test constituents were transferred into sterile test tubes and were kept in an incubator shaker for approximately 20 minutes at 37 °C and 110 rpm. After this period, soft agar containing histidine-biotin 1 tryptophan (2 mL) was added to each of the tubes.

A. For the test in the presence of metabolic activation -
a) 100 µL test concentration/solvent/appropriate positive control
b) 100 µL bacterial culture
c) 500 µL S-9 mix

B. For the test in the absence of metabolic activation -
a) 100 µL test concentration/solvent/appropriate positive control
b) 100 µL bacterial culture
c) 500 µL of PBS

The tube contents were mixed and overlaid onto VB agar plates. After the soft agar had set, the plates were incubated at 37 ± 1°C for 67 hours. After incubation, the revertant colonies in each plate were counted manually and the plates were examined for bacterial background lawn. All these procedures were carried out under yellow light.

Active ingredient analyses was performed on samples from all the dose levels along with the DMSO control.

The bacterial suspension of each tester strain was diluted up to 10-6 dilution in PBS. One hundred microlitres from the 10-6 dilution of each tester strain was
plated onto nutrient agar plates in triplicate. The plates were incubated at 37 ± 1°C for 67 hours. After incubation, the number of colonies in each plate were manually counted and expressed as the number of colony forming units per mL of the bacterial suspension.
Evaluation criteria:
The conditions necessary for determining a positive result were that there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.
For strains TA98, TA1535, and TA1537, data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean vehicle control value.
For strains TAl00 and WP2 uvrA (pKM101), data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean vehicle control value.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Statistics:
Standard statistical methods were employed
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Initial Toxicity – Mutation Assay -
The mean number of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains. For all the tester strains, the mean numbers of revertant colonies was comparable to or lower than that of the respective solvent control plates at all the tested concentrations, both in the presence and absence of metabolic activation. The test article did not precipitate the basal agar plates in any of the tested concentrations, either in the presence or in the absence of metabolic activation. The intensity of the bacterial background lawn for TA 98, TA 1535 and TA 1537 strains of Salmonella was comparable to that of the respective solvent control plates at all the tested concentrations except at 5000 µg/plate, wherein moderate thinning of the bacterial background lawn was observed both in the presence and absence of metabolic activation. At 5000 µg/plate, for Salmonella strain TA 100 and for the E. coli strain, there was a slight reduction in the bacterial background lawn in the presence of metabolic activation and a moderate reduction in the absence of metabolic activation.
Positive control chemicals tested simultaneously produced a significant increase in the mean numbers of revertant colonies for all the strains when compared to the respective solvent control plates. The intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective solvent control plates.

Confirmatory Mutation Assay -
The mean number of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains. For all the tester strains, the mean numbers of revertant colonies was comparable to or lower than that of the respective solvent control plates at all the tested concentrations, both in the presence and absence of metabolic activation. The test article did not precipitate the basal agar plates in any of the tested concentrations, either in the presence or in the absence of metabolic activation. The intensity of the bacterial background lawn for TA 98, TA 1535 and TA 1537 strains of Salmonella was comparable to that of the respective solvent control plates at all the tested concentrations except at 5000 µg/plate, wherein moderate thinning of the bacterial background lawn was observed both in the presence and absence of metabolic activation. For Salmonella strain TA 100 and for the E. coli strain, there was a slight reduction in the bacterial background lawn both in the presence and absence of metabolic activation. Positive control chemicals tested simultaneously produced a significant increase in the mean numbers of revertant colonies for all the strains when compared to the respective solvent control plates. The intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective solvent control plates.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The S-9 homogenate was found to be active and sterile and the protein content was found to be 25 mg/ml.Salmonella typhimuriumstrains TA 98, TA 100, TA 1535 and TA 1537 demonstrated the requirement of histidine amino acid for their growth. Escherichia colistrain WP2 uvrA (pKM101) demonstrated the requirement of tryptophan amino acid for its growth. Ampicillin resistance was demonstrated by the strains TA 98, TA 100 and WP2 uvrA (pKM101) which carry R-factor plasmids. The presence of characteristic mutations like the rfa mutation was demonstrated by all theSalmonella typhimuriumstrains by their sensitivity to crystal violet. The uvrA mutation in the Escherichia coJistrain and the uvrB mutation in theSalmonella typhimuriumstrains was demonstrated through their sensitivity to ultraviolet light. Finally, all these tester strains produced spontaneous revertant colonies which were within the frequency ranges of the test facility's historical control data. The test article was stable in DMSO for 24 hours at 15 and 50000 µg/ml and was homogeneous in DMSO at both the doses. The most concentrated test article dilution, the sham (PBS) and S9 mixes were found to be sterile. Viable counts of all the tester strains were within the required range of 1-2 x109CFU/ml for both the trials of the mutation assay.

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the study, UCAR n-pentyl propionate was not considered mutagenic in this bacterial reverse mutation assay at the highest tested concentration of 5000 μg/plate.
Executive summary:

UCAR n-pentyl propionate was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA 98, TA 100, TA 1535 and TA 1537 strains of Salmonella typhimurium and WP2 uvrA (pKM 101) strain of Escherichia coli in two phases. In the first phase, an initial toxicity-mutation test was performed. The second phase was an independent confirmatory mutation test. The bacterial tester strains were exposed to the test article in the presence and absence of metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver) using preincubation procedure.

UCAR n-pentyl propionate was found to be soluble in dimethyl sulphoxide (DMSO) at the required concentration of 50000 μg/mL.

In the initial toxicity-mutation test, UCAR n-pentyl propionate was exposed in duplicate at the concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate along with the solvent and appropriate positive controls. The test article did not cause any precipitation on the basal agar plates up to the highest tested concentration of 5000 μg/plate, but exhibited toxicity to the bacterial tester strains in terms of reduction in mean revertant colonies as well as thinning of bacterial background lawn at the highest tested concentration of 5000 μg/plate compared to the solvent control, in the presence and absence of metabolic activation.

Based on these initial findings, in the confirmatory mutation test, the test article was tested in triplicate at the concentrations of 100, 266, 707, 1880 and 5000 μg/plate along with the solvent and appropriate positive controls, in the presence and absence of metabolic activation. The mean and standard deviation of revertant colonies were calculated for each test concentration and the controls for all the tester strains.

The results showed that the mean numbers of revertant colonies neither doubled for strains TA 100 and WP2 uvrA (pKM101) nor tripled for strains TA 98, TA 1535 and TA 1537 when compared to the respective solvent control plates, either in the presence or in the absence of the metabolic activation at any of the tested concentrations. In this study, a significant increase in the mean numbers of revertant colonies in the positive controls was seen, demonstrating assay sensitivity.

The study indicated that the test article, UCAR n-pentyl propionate, was not mutagenic in this Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay up to the highest tested concentration of 5000 μg/plate.

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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 473, EPA OPPTS 870.5375 and 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:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - 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:
The ability of a compound to induce chromosomal damage (clastogenicity) can be assayed in vitro using cultured mammalian cells. Among various cell systems that can be used for this purpose, cultured rat lymphocytes have the following advantages. The laboratory rat is widely used for toxicological studies and hence the results of the in vitro cytogenetic tests can be compared with other toxicological endpoints. Since rats can be maintained in well-controlled environments, the influence of environmental factors on the endpoint being measured can be controlled. The karyotype of cultured rat lymphocytes (2N = 42) is stable as opposed to the relative karyotypic instability of established cell lines. Furthermore, with the rat lymphocyte system, S9 preparations from the same species can be used for metabolic activation.
Species / strain / cell type:
lymphocytes: from rats Sprague Dawley CD IGS (outbred Crl:CD(SD))
Details on mammalian cell type (if applicable):
- Type and identity of media: In the assays, 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).
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg body weight) male CD rats was purchased from Molecular Toxicology, Inc., Boone, North Carolina, and stored at -100°C or below.
Test concentrations with justification for top dose:
In a confirmatory assay performed with S9, targeted concentrations of 0 (solvent control), 300, 900, and 1442 μg/ml were selected for determining the incidence of chromosomal aberrations and incidence of polyploidy.
Vehicle / solvent:
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 DMSO 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:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C and cyclophosphamide
Details on test system and experimental conditions:
Blood samples were collected by cardiac puncture, following euthanasia with carbon dioxide. In the assays, 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.

The solvent selected for dissolving the test material (dimethyl sulfoxide (DMSO)) was used as the solvent control treatment. Mitomycin C (MMC, Sigma, St. Louis, Missouri, CAS No. 50-07-7) was used as the positive control chemical for the non-activation assay at a concentration of 0.5 μg/ml (4 hour treatment) or 0.05 and 0.075 μg/ml (24 hour treatment), while cyclophosphamide monohydrate (CP, Sigma, CAS No. 6055-19-2) was the positive control for the activation assay at final concentrations of 2 and 4 μg/ml.

S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg body weight) male CD rats was purchased from Molecular Toxicology, Inc., Boone, North Carolina, and stored at -100°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a"mix". The mix consisted of 10 mM MgCl2·6H2O (Sigma), 5 mM glucose-6-phosphate (Sigma), 4 mM nicotinamide adenine dinucleotide phosphate (Sigma), 10 mM CaCl2 (Fisher, Fair Lawn, New Jersey), 30 mM KCl (Sigma), and 50 mM sodium phosphate (pH 8.0, Sigma and Fisher). 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 was 1/5 of the concentrations.

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 DMSO and further diluted (1:100) with the treatment media to obtain the desired concentrations. All dosing units were expressed in μg/ml. MMC and CP were dissolved in RPMI 1640 with HEPES and antibiotics.

The selected concentrations of the test material were diluted in an appropriate solvent and analyzed by gas chromatography with flame ionization detection (GC/FID). All test cultures were identified using self adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

The cultures were treated with various concentrations of the test material and the selected concentration of the positive control chemicals. The highest concentration of the test material assayed was based on a limit concentration of 10 mM in this assay system. The other concentrations tested were separated by a factor of 2. 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. Mitotic indices were determined as the number of cells in metaphase among
1000 cells/replicate and expressed as percentages.

Initially, slides from the short treatment (with and without S9) were evaluated for cytogenetic analysis. Slides from the continuous treatment without S9 were evaluated only when the results with short treatment yielded negative findings. Slides from the solvent controls, positive controls, and three concentrations of the test material were selected for cytogenetic analysis.

One hundred metaphases/replicate were examined from coded slides for structural abnormalities. The number of cells examined for structural abnormalities was reduced to 50 metaphases per replicate when high numbers of aberrations were observed (approximately 20%). The microscopic coordinates of metaphases containing aberrations were recorded. Only those metaphases that contained 42 + 2 centromeres were scored with the exception of cells with multiple aberrations, in which case accurate counts of the centromeres would not be possible. Structural chromosomal abnormalities counted include chromatid and chromosome gaps, chromatid breaks and exchanges, chromosome breaks and exchanges, and miscellaneous (chromosomal disintegration, chromosomal pulverization, etc.). Those cells having five or more aberrations/cell were classified as a cell with multiple aberrations. Chromatid gaps and chromosome gaps were not included in calculations of total cytogenetic aberrations. In addition, a total of 100 metaphases/replicate were examined for incidence of polyploidy.
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 will be considered positive in this assay if it induces a significant, dose-related increase in the frequency of cells with aberrations and the incident of aberrant cells and cultures is outside the recent historical solvent control range.
Statistics:
The proportions of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two-way contingency table was constructed to analyze the frequencies of aberrant cells. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the global hypothesis of no difference in the average number of cells with aberrations among the dose groups. An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If this statistic was found to be significant at alpha = 0.05, versus a one-sided increasing alternative, pairwise tests (i.e. control vs. treatment) were performed at each dose level and evaluated at alpha = 0.05, again versus a one-sided alternative. If any of the pairwise tests were significant, a test for linear trend of increasing number of cells with aberrations with increasing dose was performed.

Polyploid cells were analyzed by the Fisher Exact probability test. The number of polyploid cells were pooled across replicates for the analysis and evaluated at alpha = 0.05. The data was analyzed separately based on the presence or absence of S9 and based on the exposure time.
Species / strain:
lymphocytes:
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:
Assay A1
In the initial 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), 22.6, 45.1, 90.2, 180.3, 360.5, 721.0, and 1442.0 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 11.3 μg/ml. Analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 91.1 to 117.5% of the target and these values also indicated stability of the test material
in the solvent.

Short Treatment (4 hour treatment) -
In the absence of S9, the cultures displayed excessive toxicity at the two highest concentrations (721.0 and 1442.0 μg/ml) while the remaining concentrations displayed moderate to no toxicity with relative mitotic indices ranging from 80.1 to 102.9% compared to the solvent control value. Based upon these results, it was determined that the appropriate level of cytotoxicity was not achieved in the absence of S9 with a 4 hour treatment; therefore, this portion of the assay was repeated as Assay B1.

In the presence of S9, relative mitotic indices of the treated cultures ranged from 54.8 to 99.1% as compared to the solvent control value. Based upon these results, cultures treated with targeted concentrations of 0 (solvent control), 360.5, 721.0, and 1442.0 μg/ml were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy in the presence of S9 activation. Among the cultures treated with the positive control chemical, 4 μg/ml of CP was selected for evaluation of aberrations in the presence 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 activation assay, cultures treated with the test material at concentrations of 360.5, 721.0, and 1442.0 μg/ml had aberrant cell frequencies of 1.0, 1.5 and 2.5%, respectively as compared to the solvent control value of 0.5%. Statistical analyses of these data did not identify significant differences between the solvent control and any of the treated cultures. The frequencies of aberrant cells observed in the test material treated cultures 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 chemical. Aberrant cell frequency in CP cultures was 17.3%.

Continuous Treatment (24 hour treatment) -
Cultures treated continuously for 24 hours in the absence of S9 activation had excessive toxicity at the highest concentrations (721.0 and 1442.0 μg/ml), while the remaining cultures had relative mitotic indices ranging from 45.3 to 96.2% relative to the solvent control value. Due to the inability to evaluate the 4 hour test material treated cultures in the absence of S9, the 24 hour slides were not evaluated for chromosomal aberrations or polyploidy until results were obtained from the 4 hour treatment.

Assay B1
This assay was conducted to 1) repeat the short 4 hour treatment in the absence of S9 since the desired level of toxicity was not achieved in Assay A1, and 2) confirm the results obtained in the presence of S9 in Assay A1. Cultures were treated with the test material in the absence of S9 activation for 4 hours at concentrations of 0 (solvent control), 50, 100, 200, 300, 400, 500, 600, and 700 μg/ml and presence of S9 at 0 (solvent control), 100, 300, 500, 700, and 1442 μg/ml.

In the absence of S9, the cultures displayed toxicity at the three highest concentrations (500, 600, and 700 μg/ml) with the remaining concentrations having relative mitotic indices ranging from 28.7 to 107.0% compared to the solvent control value. In the presence of S9, cultures displayed toxicity excessive toxicity at the highest concentration with no observable mitotic figures. The remaining concentrations had relative mitotic indices ranging from 74.8 to 100.8% as compared to the solvent control value. Based upon these results, it was determined that the appropriate level of cytotoxicity was not achieved in the absence or the presence of S9 with a 4 hour treatment; therefore, this assay was repeated as Assay C1.

Assay C1
In the repeat assay, cultures were treated in the absence of S9 for 4 hours with targeted concentrations of 0 (solvent control), 100, 200, 250, 300, 350, 400, 450, 500, and 600
μg/ml and in the presence of S9 for 4 hours at targeted concentrations of 0 (solvent control), 300, 500, 700, 800, 900, 1000, 1100, 1200, 1300, and 1442 μg/ml.

In the absence of S9, the cultures displayed excessive toxicity at the highest concentration (600 μg/ml) while the remaining concentrations displayed moderate to no toxicity with relative mitotic indices ranging from 26.4 to 80.8% compared to the solvent control value. In the presence of S9, relative mitotic indices of the treated cultures ranged from 47.5 to 103.0% as compared to the solvent control value. Based upon these results, cultures treated with targeted concentrations of 0 (solvent control), 250, 350, and 450 μg/ml in the absence of S9 and 0 (solvent control), 300, 900, and 1442 μg/ml in the presence of S9 were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy. Among the cultures treated with the positive control chemicals for 4 hours, 0.5 μg/ml MMC (absence of S9) and 4 μg/ml of CP (presence of S9) were selected for evaluation of aberrations.

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 absence of S9, the frequency of cells with aberrations in the solvent control was 0.5% and the corresponding values at concentration levels of 250, 350, and 450 μg/ml were 1.0, 1.5, and 0.5%, respectively. In the presence of S9, cultures treated with the test material at concentrations of 300, 900, and 1442 μg/ml had aberrant cell frequencies of 0.0, 1.0, and 0.5%, respectively as compared to the solvent control value of 0.5%. Statistical analyses of these data did not identify significant differences between the solvent control and any of the treated cultures in the absence or presence of S9. The frequencies of aberrant cells observed in the test material treated cultures 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 frequency in MMC treated cultures (-S9, 4 hour treatment) was 46.0% and in CP treated cultures (+S9, 4 hour treatment) was 16.0%.

Continuous Treatment in the Absence of S9 (24 hour treatment, Assay A1) -
Based upon the negative finding in the 4 hour treatment in the absence of S9 from Assay C1, cultures treated continuously in Assay A1 were evaluated at targeted concentrations of 0 (solvent control), 45.1, 90.2, and 360.5 μg/ml for the determination of chromosomal aberration frequencies and incidence of polyploidy. Cultures treated with 0.075 μg/ml MMC were selected to serve as the positive control.

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

The frequency of aberrant cells in the solvent control was 1.5% and the corresponding values at concentration levels of 45.1, 90.2, and 360.5 μg/ml were 1.5, 1.0, and 1.0%, 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.

A significant increase in the frequency of cells with aberrations was observed in cultures treated with the positive control chemical. Aberrant cell frequency in MMC treated cultures was 35.0%.
Remarks on result:
other: other:
Remarks:
Migrated from field 'Test system'.

The pH and osmolality of treatment medium containing approximately 1754 μg UCAR n-Pentyl Propionate/ml (approximately 10 mM limit) and medium containing 1% solvent (DMSO) was determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts), respectively. There was no appreciable or unacceptable 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 = 7.50, osmolality = 449 mOsm/kg H20; culture medium with 1% DMSO, pH = 7.51, osmolality = 481 mOsm/kg H20).

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions used, UCAR n-pentyl propionate was non-genotoxic in this in vitro chromosomal aberration test.
Executive summary:

UCAR n-Pentyl Propionate (pentyl ester of propanoic acid) 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 1442 μg UCAR n-Pentyl Propionate per ml of culture medium. The duration of treatment was 4 or 24 hours without S9 and 4 hours with S9. The highest concentration was based on the limit concentration of 10 mM in this assay system. Based upon the mitotic indices, cultures treated for 4 hours with targeted concentrations of 0 (solvent control), 250, 350, and 450 μg/ml in the absence of S9 activation and 0 (solvent control), 360.5, 721, and 1442 μg/ml in the presence of S9 activation were selected for determining the incidence of chromosomal aberrations and incidence of polyploidy. Cultures treated for 24 hours in the absence of S9 with targeted concentrations of 0 (solvent control), 45.1, 90.2, and 360.5 μg/ml were selected for determining the incidence of chromosomal aberrations. In a confirmatory assay performed with S9, targeted concentrations of 0 (solvent control), 300, 900, and 1442 μg/ml were selected for determining the incidence of chromosomal aberrations and incidence of polyploidy. There were no statistically significant increases in the frequencies of cells with aberrations in any of the treatments in either the absence or presence 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, UCAR n-Pentyl 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:
experimental study
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).
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: other: CHO-K1, (ATCC CCL-61, Lot 4765275)
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

In a GLP study conducted according to OECD TG 471 (Bacterial Reverse Mutation Assay), UCAR n-pentyl propionate was not considered mutagenic at the highest tested concentration of 5000 μg/plate. In a second GLP study conducted according to OECD TG 473 (In vitro Mammalian Chromosome Aberration Test), UCAR n-pentyl propionate was not genotoxic. In a third GLP study, conducted according to OECD TG 476 (In vitro Mammalian Cell Gene Mutation Test) there was no evidence of genotoxicity at the highest, non-cytotoxic, concentrations in the presence or absence of metabolic activation.

Justification for classification or non-classification

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