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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
bacterial reverse mutation assay (e.g. Ames test)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 December 2015 to 11 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
other: yellow semi-solid
Specific details on test material used for the study:
- Purity: 100% (UVCB)
- Physical state/Appearance: Yellow semi-solid
- Expiry Date: 23 November 2017
- Storage Conditions: Room temperature in the dark

No correction was made for purity.

The test item was insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully soluble in dimethyl formamide at the same concentration and acetone at 100 mg/mL in solubility checks performed in-house. Acetone was selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer and sonication for 10 mins at 40 °C on the day of each experiment. No correction was made for purity. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1: Range-finding test: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: Main test: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The substance was not sufficiently soluble in water and dimethyl sulphoxide but was soluble in acetone.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Concurrent - Acetone)
True negative controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2, 3, 5 µg/plate respectively for WP2uvrA, TA100, TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Concurrent - Acetone)
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537
Untreated negative controls:
no
Remarks:
(Concurrent - Acetone)
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Concurrent - Acetone)
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
1, 2, 10 µg/plate for TA100, TA1535&TA1537, WP2uvrA respectively
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Concurrent - Acetone)
True negative controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) at multiple dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard/sterilized co-factors).

EXPRIMENT 1
Dose selection
- The maximum concentration was 5000 µg/plate (the maximum recommended dose level).
-Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
-0.1 mL of the appropriate concentration of test item, solvent vehicle, or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate.
- Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described above except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 °C+/- 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
-A number of manual counts were performed due to colonies spreading and bubble interference, disturbing the actual plate count.

EXPERIMENT 2
Dose selection
The dose range used for experiment 2 was determined by the results of experiment 1 and was 5 to 5000 µg/plate.
7 test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item.

Without Metabolic Activation
The procedure was the same as described previously.

With Metabolic Activation
The procedure was the same as described previously.

Incubation and Scoring
All of the plates were incubated at 37 °C +/- 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were performed for count verification purposes due to toxicity.

DURATION
- Preincubation period: N/A
- Exposure duration: Approximately 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A


SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9

NUMBER OF CELLS EVALUATED:
All strains: 0.9 to 9 *10^9 bacteria/mL

DETERMINATION OF CYTOTOXICITY
- Method: N/A

OTHER EXAMINATIONS:
N/A


OTHER:
Acceptance Criteria
All bacterial strains must have demonstrated the required characteristics as determined by their
respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).

All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535: 7 to 40
TA100: 60 to 200
TA1537: 2 to 30
TA98: 8 to 60
WP2uvrA: 10 to 60

Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All
of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.

There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1 . A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for TA 1535, TA1537 and TA98 at 5000 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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

Any other information on results incl. tables

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate.The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of S9-mix, initially from 1500 μg/plate (TA1537) and at 5000 μg/plate (remaining tester strains). In the presence of S9-mix, weakened bacterial background lawns were noted to TA1535, TA98 and TA1537 at 5000 μg/plate. No toxicity was noted to either TA100 or WP2uvrA. Consequently the same maximum dose level (5000 μg/plate) was employed in the second mutation test. A similar toxic response was noted in Experiment 2 with weakened bacterial background lawns noted at 5000 μg/plate to all of the tester strains dosed in the absence of S9-mix.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in experiment 1. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in experiment 2.

 

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

INTRODUCTION

The method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline – Bacterial Reverse Mutation Test.

 

METHODS

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to 8 dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate.

 

Seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.

 

RESULTS

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of S9-mix, initially from 1500 µg/plate (TA1537) and at 5000 µg/plate(remaining tester strains). In the presence of S9-mix, weakened bacterial background lawns were noted to TA1535, TA98 and TA1537 at 5000 μg/plate. No toxicity was noted to either TA100 or WP2uvrA. Consequently the same maximum dose level (5000 μg/plate) was employed in the second mutation test. A similar toxic response was noted in Experiment 2 with weakened bacterial background lawns noted at 5000 μg/plate to all of the tester strains dosed in the absence of S9-mix. There was no visible reduction in the growth of the bacterial background lawns of any tester strain dosed in the presence of S9-mix, however small reductions in TA1535 and TA98 revertant colony frequency were noted at the maximum dose level. A light test item film was noted at 5000 μg/plate; this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in experiment 1. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in experiment 2.

 

CONCLUSION

The test substance was considered to be non-mutagenic under the conditions of this test.