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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 June 2019 to 11 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
31 March 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register.
Version / remarks:
77:33748-33749
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Light yellow liquid
- Storage conditions: Ambient temperature

Method

Target gene:
Histidine and tryptophan
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Moltox post mitochondrial supernatant (S9)
Test concentrations with justification for top dose:
- Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Experiment 2: 15, 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
Acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone (Acros Organics; batch 1882926; purity ≥ 99.75 %; expiry date July 2020)
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 (all without S9)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 μg/plate for TA1537 without S9
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 μg/plate for TA98 without S9
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2uvrA (all with S9)
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 μg/plate for TA98 with S9
Details on test system and experimental conditions:
STUDY CONTROLS
- The vehicle control used was acetone. The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.
- The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.

STERILITY CONTROLS
- Top agar and histidine/biotin or tryptophan in the absence of S9-mix (in triplicate).
- Top agar and histidine/biotin or tryptophan in the presence of S9-mix (in triplicate).
- The maximum dosing solution of the test item in the absence of S9-mix only (test in singular prior to Experiment 1).

MICROSOMAL ENZYME FRACTION
- The S9 Microsomal fractions (Sprague-Dawley) were purchased from Moltox.
- Lot No. PB/βNF S9 4061 was used in this study.
- A copy of the S9 Certificate of Efficacy is presented in Appendix 3 (attached).

S9 MIX AND AGAR
- The S9-mix was prepared before use using sterilised co-factors and maintained on ice for the duration of the test.
- The S9 mix contained 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.

MEDIA
- Top agar was prepared using 0.6 % Bacto agar (lot numbers 7193746 04/2022 and 8255817 07/2023) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar.
- Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 51159 07/2019 and 51213 08/2019).

BACTERIA
- The five strains of bacteria used are shown in the table below together with their mutations.
- All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
- The bacteria used in the test were obtained from British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987 or 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.
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 2321478 04/2023) 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.

TEST ITEM PREPARATION AND ANALYSIS
- The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
- The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in pre-dried acetone by mixing on a vortex mixer and sonication for 5 minutes at 40 °C. No correction for purity was required.
- 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.

DOSE SELECTION FOR EXPERIMENT 1
- 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.

EXPERIMENT 1 - WITHOUT METABOLIC ACTIVATION
- An aliquot (0.1 mL) of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. 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.

EXPERIMENT 1 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously except that following addition of the test item formulation and bacterial culture, S9-mix (0.5 mL) was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

EXPERIMENT 1 - INCUBATION AND SCORING
- 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.
- The plates were viewed microscopically for evidence of thinning (toxicity).

DOSE SELECTION FOR EXPERIMENT 2
- Following Sponsor recommendation the plate incorporation method was employed for Experiment 2 in the presence and absence of metabolic activation (S9-mix).
- The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 μg/plate.
- Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity of the test item.

EXPERIMENT 2 - WITHOUT METABOLIC ACTIVATION
- The procedure was the same as described previously.

EXPERIMENT 2 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously.

EXPERIMENT 2 - INCUBATION AND SCORING
- 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.
- The plates were viewed microscopically for evidence of thinning (toxicity).
- Sporadic manual counts were performed due to spreading colonies and light background contamination which prevented an accurate automated count.

ACCEPTANCE CRITERIA
- The reverse mutation assay may be considered valid if the following criteria are met:
(i) 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), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
(ii) 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 TA 1535 (7 to 40); TA100 (60 to 200); TA1537 (2 to 30); TA98 (8 to 60); WP2uvrA (10 to 60). Combined historical negative and solvent control ranges for 2017 and 2018 are presented in Appendix 2 (attached).
(iii) All tester strain cultures should be in the range of 0.9 to 9 x 10E+09 bacteria per mL.
(iv) 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. The historical ranges of the positive control reference items for 2017 and 2018 are presented in Appendix 2 (attached).
(v) There should be a minimum of four non-toxic test item dose levels.
(vi) There should be no evidence of excessive contamination.

MAJOR COMPUTERISED SYSTEMS
- Perceptive Instruments – Ames Study Manager v1.24 and Ames Sorcerer v3.
Rationale for test conditions:
- The purpose of the study was to evaluate the test item for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.
- The study was based on the in vitro technique described by Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. The Salmonella typhimurium strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base-pair substitution to tryptophan independence (Green and Muriel, 1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.
- Since many compounds do not exert a mutagenic effect until they have been metabolized by enzyme systems not available in the bacterial cell, the test item and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre-treated with a mixture known to induce an elevated level of these enzymes.
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:
(i) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
(ii) A reproducible increase at one or more concentrations.
(iii) Biological relevance against in-house historical control ranges.
(iv) 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).
(v) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- 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 significance was confirmed by using Dunnetts 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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
- Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (attached) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
- 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation (S9-mix), are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2. The results are also expressed graphically in Figure 1 to Figure 4. All relevant tables and figures are attached.
- A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 2 (attached).

EXPERIMENT 1
- The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate.
- 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).
- A test item precipitate (globular in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix). 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).

EXPERIMENT 2
- The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 μg/plate).
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).
A test item precipitate (globular in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix). 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).

Applicant's summary and conclusion

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

GUIDELINE

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", 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 the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and 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 15 to 5000 μg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity 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 and 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 OECD TG 471 recommended dose level of 5000 μg/plate. 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), 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 (plate incorporation method). Similarly, 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).

 

A test item precipitate (globular in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. 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 (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 (plate incorporation method).

 

CONCLUSION

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