Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 July 2016 to 12 August 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 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
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Extremely pale yellow liquid
- Storage conditions: Room temperature in the dark.

Method

Target gene:
Histidine and tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital / β-naphtha flavone induced 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
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
ENNG (CAS number 4245-77-6; Batch 67F-3700; Purity not available; Expiry date 18 September 2017; Solvent DMSO)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
9AA (CAS number 90-45-9; Batch S32398-438; Purity 99.9 %; Expiry date 01 October 2017; solvent DMSO)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4NQO (CAS number 56-57-5; Batch 030M1206; Purity 100 %; Expiry date 08 October 2017; Solvent DMSO)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-Aminoanthracene
Remarks:
2AA (Cas number 613-13-8; Batch STBB1901M9; Purity 97.5 %; Expiry date 08 October 2017; Solvent DMSO)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
benzo(a)pyrene
Remarks:
BP (CAS number 50-32-8; Batch 090M1400V; Purity 96 %; Expiry date 12 October 2017; Solvent DMSO)
Details on test system and experimental conditions:
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 only).

MICROSOMAL ENZYME FRACTION
- The S9 Microsomal fractions were pre-prepared using standardised in-house procedures (outside the confines of this study).
- Lot No. PB/PNF S9 10 April 2016 was used for Experiment 1 and Lot No. PB/PNF S9 05 June 2016 was used for Experiment 2.
- Copies of the S9 Certificates of Efficacy are 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.
- 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.
- 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 was added to each 100 mL of top agar.
- Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (Lot numbers 41961 07/16 and 42228 09/16).

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 L T2 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 TAI 00~ the R-factor plasmid pKMIOI 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 auvrA-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 the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on a nutrient agar plate, on 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 number 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
- In 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 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 i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10E-04 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 was 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 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.

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, 0.5 mL of S9-mix 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).

DOSE SELECTION FOR EXPERIMENT 2
- 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 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 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).

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) and Mortelmans and Zeiger (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 2012 and 2013 are presented in Appendix 1 (attached).
(iii) All tester strain cultures should be in the range of 0.9 to 9 x 10E09 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 2012 and 2013 are presented in Appendix 1 (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
- Ames Study Manager and Sorcerer Imaging System.
- Delta Building Monitoring System.
Evaluation criteria:
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) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
(v) 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
- 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 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
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
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
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
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 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 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 Table 2 and Table 3 for Experiment 1 (attached) and Table 4 and Table 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. 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. A test item precipitate (globular in appearance) was noted under a low power microscope at 150 μg/plate and by eye from 500 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 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 TA98 revertant colony frequency were observed in the second mutation test at and above 1500 μg/plate (presence of S9-mix only). These increases were 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 1.5 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 study.
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 typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere 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 repeatedon 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. A test item precipitate (globular in appearance) was noted under a low power microscope at 150 μg/plate and by eye from 500 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 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 TA98 revertant colony frequency were observed in the second mutation test at and above 1500 μg/plate (presence of S9-mix only). These increases were 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 1.5 times the concurrent vehicle control.

 

CONCLUSION

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