Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD 471 and under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
his gene
Species / strain
Species / strain / cell type:
other: TA1535, TA1537, TA98, TA100 and WP2uvrA
Additional strain / cell type characteristics:
other: rfa: deep rough (defective lipopolysaccharide cellcoat) gal: mutation in the galactose metabolism chl: mutation in nitrate reductase bio: defective biotin synthesis uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg).
Test concentrations with justification for top dose:
17 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in all five tester strains. The test substance precipitated on the plates at dose levels of 1600 and 5000 μg/plate. In a follow-up experiment of the assay with additional parameters, the test substance was tested at a concentration range of 154 to 1568 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains
Vehicle / solvent:
dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany)
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Controls -S9 TA1535: sodium azide, TA1537: ICR-191, TA98: 2-nitrofluorene, TA100: methylmethanesulfonate, WP2uvrA: 4-nitroquinoline N-oxide Controls +S9 2-aminoanthracene for all tester strains
Details on test system and experimental conditions:
Ascorbyl palmitate was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).
Ascorbyl palmitate was dissolved in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany). In the dose range finding study, the stock solution of 50 mg/ml was dissolved by vortexing only. In the mutation assays, the stock solution was treated with ultrasonic waves until the test substance had completely dissolved. Test substance concentrations were prepared directly prior to use and used within 2 hours after preparation.
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of Ascorbyl palmitate used in the subsequent mutation assay was 5000 µg/plate or the level at which the test substance exhibited limited solubility.
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. The above mentioned dose range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test substance was tested both in the absence and presence of 5% (v/v)
S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:
according to OECD Guideline 471
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
no formal hypothesis testing was done

Results and discussion

Test results
Species / strain:
other: four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

In the dose range finding test, the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Ascorbyl palmitate precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested. Since Ascorbyl palmitate precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined. Cytotoxicity, as evidenced by slight to moderate decreases in the number of revertants, was only observed in tester strain TA100 in the absence of S9-mix at the test substance concentrations of 512 and 1600 µg/plate. Results of this dose range finding test were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 17 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test substance precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested. Since Ascorbyl palmitate precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined. No biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test substance was tested at a concentration range of 154 to 1568 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance precipitated on the plates at the dose level of 1568 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Ascorbyl palmitate did not induce a biologically significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative control values were not all within the laboratory historical control data ranges, specifically TA1537 (first experiment, absence of S9-mix). However, this response was judged not to impact the reliability of the study. The deviation in the negative control value was minor, the value (2) was just below the limit of the range (3).

The strain-specific positive control substances were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:
negative in the absence of metabolic activation
negative in the presence of metabolic activation

Ascorbyl palmitate is not mutagenic in the Ames Test
Executive summary:

Ascorbyl palmitate was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). Each concentration was tested in triplicate. The positive and negative controls produced the expected results.

Based on the results of this study it is concluded that Ascorbyl palmitate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.