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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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).
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): n-propyl propionate
- Physical state: transparent, colourless liquid
- Analytical purity: 99.96% (by area normalization gas chromatography)
- Lot/batch No.: Lot No.: QC1355VlCl
- Storage condition of test material: room temperature

Method

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
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
Controls
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

Results and discussion

Test results
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'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

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).