[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 June 2016 to 24 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

S9 fraction pre-prepared in bulk with no impact on validity or integrity of study (see below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

S9 fraction pre-prepared in bulk with no impact on validity or integrity of study (see below)

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

yes

Remarks:

S9 fraction pre-prepared in bulk with no impact on validity or integrity of study (see below)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: OCSPP harmonized guideline - Bacterial reverse mutation assay

Deviations:

yes

Remarks:

S9 fraction pre-prepared in bulk with no impact on validity or integrity of study (see below)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Appearance/physical state: Colourless to yellow liquid
- Storage conditions: Room temperature in the dark

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

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

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Controls 2

Untreated negative controls:

yes

Remarks:

untreated controls

Controls 3

Positive controls:

yes

Remarks:

without metabolic activation (2 µg/plate for WP2uvrA; 3 µg/plate for TA100; 5 µg/plate for TA1535)

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

ENNG; direct acting compound; batch 67F-3700 treated as 100 % despite no data sheet being available; expiry date 18 September 2017; solvent DMSO

Controls 4

Positive controls:

yes

Remarks:

without metabolic activation (80 µg/plate for TA1537)

Positive control substance:

9-aminoacridine

Remarks:

9AA; direct acting compound; batch S32398-438; purity 99.9 %; expiry date 01 October 2017; solvent DMSO

Controls 5

Positive controls:

yes

Remarks:

without metabolic activation (0.2 µg/plate for TA98)

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

4NQO; direct acting compound; batch 030M1206; purity 100 %; expiry date 08 October 2017; solvent DMSO

Controls 6

Positive controls:

yes

Remarks:

with metabolic activation (1 µg/plate for TA100; 2 µg/plate for TA1535 and TA1537; 10 µg/plate for WP2uvrA)

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA; indirect acting compound; batch STBB1901M9; purity 97.5 %; expiry date 08 October 2017; solvent DMSO

Controls 7

Positive controls:

yes

Remarks:

with metabolic activation (5 µg/plate for TA98)

Positive control substance:

benzo(a)pyrene

Remarks:

BP; indirect acting compound; batch 090M1400V; purity 96 %; solvent DMSO

Details on test system and experimental conditions:

STUDY CONTROLS
- Acetone was used as the solvent (vehicle) control.
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
- 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.
- Positive controls were performed in triplicate.
- Sterility controls were performed (i) top agar and histidine/biotin or tryptophan in the absence of S9 mix in triplicate (ii) top agar and histidine/biotin or tryptophan in the presence of S9 mix in triplicate (iii) singular test of maximum dosing solution of the test item in the absence of S9 mix only.

MICROSOMAL ENZYME FRACTION
- The S9 Microsomal fractions were pre-prepared using standardized in-house procedures.
- Lot No. PB/βNF S9 10 April 2016 was used in the study.
- A copy of the S9 Certificate of Efficacy is presented in Appendix 2 (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.
- Composition of the S9-mix was 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) and sterile
distilled water (14.5 mL).
- An aliquot (0.5 mL) of S9-mix and molten, trace histidine or tryptophan supplemented, top agar (2 mL)
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 the experiment.

MEDIA
- Top agar was prepared using 0.6% Bacto agar (lot number 5054857 12/19) 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 41720 06/16 and 41792 07/16).

BACTERIA
- Details of the five strains of bacteria used and their mutations are attached.
- 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).
- Bacteria used in the test were obtained from University of California, Berkeley, on culture discs (04 August 1995) and British Industrial Biological Research Association, on a nutrient agar plate (17 August 1987).
- 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 numbers 1712138 07/20 and 1758279 10/20) and incubated at 37 °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
- During solubility checks performed in-house the test item was noted to be immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully soluble in acetone at the same concentration in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
- The test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. No correction was made for purity.
- Prior to use, the solvent was dried to remove water using molecular sieves (2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns).
- 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.

EXPERIMENT 1 – DOSE SELECTION
- 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.
- The direct plate incorporation method was used.

EXPERIMENT 1 – WITHOUT METABOLIC ACTIVATION
- An aliquot (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.
- The components were mixed and overlaid onto a Vogel-Bonner agar plate.
- Negative (untreated) controls were also performed on the same day as the mutation test.
- Each concentration of the item, appropriate positive, vehicle and negative controls was assayed in triplicate for each bacterial strain.

EXPERIMENT 1 – WITH METABOLIC ACTIVATION
- The procedure was the same as described previously except that, following the addition of 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 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 required, predominantly due to interference in the base agar (such as minor precipitation of salts/dust particles slightly distorting the counts).

EXPERIMENT 2 – DOSE SELECTION
- The procedure was repeated using the plate incorporation method in the presence and absence of metabolic activation.
- The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate.
- Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item.

EXPERIMENT 2 – WITHOUT METABOLIC ACTIVATION
- The procedure was the same as described for Experiment 1.

EXPERIMENT 2 – WITH METABOLIC ACTIVATION
- The procedure was the same as described for Experiment 1.

EXPERIMENT 2 – INCUBATION AND SCORING
- All of the plates were incubated at 37 ± 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).

ACCEPTABILITY CRITERIA
- The reverse mutation assay may be considered valid if the criteria given below are met.
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks (Ames et al, 1975; Maron and Ames, 1983; 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 7 to 40 (TA1535); 60 to 200 (TA100); 2 to 30 (TA1537); 8 to 60 (TA98); 10 to 60 (WP2uvrA).
- Combined historical negative and solvent control ranges for 2014 and 2015 are presented in Appendix 1 (attached).
- All tester strain cultures should be in the range of 0.9 to 9 x 10E09 bacteria per mL.
- 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 (with and without metabolic activation). The historical ranges of the positive control reference items for 2014 and 2015 are presented in Appendix 1 (attached).
- There should be a minimum of four non-toxic test item dose levels.
- There should be no evidence of excessive contamination.

MAJOR COMPUTERISED SYSTEMS
- Ames Study Manager and Sorcerer Imaging System.
- Delta Building Monitoring System.

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:
(a) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
(b) A reproducible increase at one or more concentrations.
(c) Biological relevance against in-house historical control ranges.
(d) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
(e) 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 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 results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 3

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

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

Additional information on results:

MUTATION TEST
- 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 were 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 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, are presented in Tables 2 and 3 (attached) for Experiment 1 and Table 4 and 5 for Experiment 2 (attached).
- A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1 (attached).
- The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.
- In the first mutation test, there was no visible reduction in the growth of the bacterial background lawn a t any dose level, either in the presence or absence of metabolic activation (S9-mix). The same maximum dose level was therefore used in the second mutation test.
- 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), in the second mutation test.
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- In Experiment 1, 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).
- Similarly, no toxicologically 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.
- Small, statistically significant increases in TA1537 revertant colony frequency were observed in the second mutation test at 5000 μg/plate (absence of S9-mix only). This increase was considered to be of no biological relevance because there was no evidence of reproducibility or a dose-response relationship. Furthermore, the individual revertant counts at the statistically significant dose level were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.6 times the
concurrent vehicle control.
- 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 the 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 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 atup to eight dose levels, in triplicate, both with and without the addition of a rat liverhomogenate metabolizing system (10% liver S9 in standard co-factors). The dose range forExperiment 1 was predetermined and was 1.5 to 5000μg/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 15 to 5000 μg/plate. Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential 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. 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 and consequently the same maximum dose level was used in the second mutation test. 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), in the second mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or 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 toxicologically 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. Small, statistically significant increases in TA1537 revertant colony frequency were observed in the second mutation test at 5000 μg/plate (absence of S9-mix only). This increase was considered to be of no biological relevance because there was no evidence of reproducibility or a dose-response relationship. Furthermore, the individual revertant counts at the statistically significant dose level were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.6 times the concurrent vehicle control.

 

CONCLUSION

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