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Genetic toxicity in vitro

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Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date - 25 November 2020; Experimental completion date - 14 December 2020; Study completion date -
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Identification: 4-Hydroxycoumarin
Physical state/Appearance: Beige powder
CAS Number: 1076-38-6
Batch Number: HCN20002
Purity: > 99%
Expiry Date: 26 September 2022
Storage Conditions: Room temperature in the dark.
Target gene:
histidine or tryptophan locus
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):
CELLS USED
The bacteria used in the test were obtained from:
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
- Trinova Biochem GmbH on 27 June 2017
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. On a regular basis (approximately monthly), batches of culture from master stocks are prepared and coded, these are then routinely tested for appropriate characteristics, viability and mutation frequency to ensure acceptability criteria is met.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 2928960 expiry date 02/2025) and incubated at 37 ± 3 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
Metabolic activation:
with and without
Metabolic activation system:
Microsomal Enzyme Fraction:
The Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley) used in this study were purchased from Moltox; Lot No. 4272 and the protein level was adjusted to 20 mg/mL.

S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Test concentrations with justification for top dose:
Experiment I - Plate incorporation method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment II: Pre-incubation method: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
DImethyl sulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2 µg/plate for WP2uvrA; 3 µg/plate for TA100; 5 µg/plate for TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
1 µg/plate for TA100; 2 µg/plate for TA1535 and TA1537; 10 µg/plate for WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation) in experiment I; preincubation in experiment II

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes
- Exposure duration/duration of treatment: All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition.

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Increase in mutant frequency.
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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
Statistics:
Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See "Additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See "Additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See "Additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See "Additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See "Additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. A single count for TA1535 (vehicle control dosed in the presence of S9-mix after the second mutation test) was just below the minimum level of the in-house historical untreated/vehicle control minima for the tester strain. This count was considered acceptable as the other vehicle and untreated control counts were within the expected range and the tester strain responded very well to the respective positive controls in both the presence and absence of S9 mix. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1 (plate incorporation):
The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of metabolic activation (S9-mix) from 1500 µg/plate to TA100, TA1535 and TA1537 and at 5000 µg/plate to TA98 and WP2uvrA. In the presence of metabolic activation, toxicity was noted from 1500 µg/plate to WP2uvrA and at 5000 µg/plate to TA98, TA100, TA1535 and TA1537.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (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).

Experiment 2 (pre-incubation):
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix) after employing a 20 minute pre-incubation modification at 37 °C.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (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).
Conclusions:
The test substance did not induce an increase in the frequency of revertant colonies, either with or without metabolic activation (S9-mix). Under the conditions of this test 4-Hydroxycoumarin was considered to be non-mutagenic.
Executive summary:

Introduction

The test 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”, 21 July 1997 as corrected in 2020, Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) 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 both the Ames plate incorporation and pre-incubation methods at up to nine 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 (plate incorporation) was 1.5 to 5000 µg/plate.  The experiment was repeated on a separate day (pre-incubation method) 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 0.5 to 5000 µg/plate. Nine test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non toxic dose levels as required by the test guideline, and were selected based on the cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Results

The vehicle (dimethyl sulphoxide) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 1500 µg/plate in both the presence and absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method). There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix) after employing a 20 minute pre-incubation procedure.  

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

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 (plate incorporation method).  

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 (pre incubation method).  

Conclusion

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item 4-Hydroxycoumarin did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix).  Under the conditions of this test 4-Hydroxycoumarin was considered to be non-mutagenic.

Additional information

Justification for classification or non-classification