Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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 19 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:
other: EPA OCSPP harmonised guideline - Bacterial Reverse Mutation Test
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: Light brown, slightly viscous, liquid
- Storage conditions: Room temperature in the dark
Specific details on test material used for the study:
- Formulated concentrations were adjusted to allow for the stated water/impurity content (41 %) of the test item

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:
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:
- Sterile distilled water
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
Untreated negative controls:
yes
Remarks:
untreated controls
Positive controls:
yes
Remarks:
without metabolic activation (2 µg/plate for WP2uvrA; 3 µg/plate for TA100 and 5 µg/plate for TA1535)
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
ENNG; direct acting compound; batch 67F-3700; expiry date 18 September 2017; solvent DMSO
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
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 56-57-5; purity 100 %; expiry date 08 October 2017; solvent DMSO
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 613-13-8; purity 97.5 %; expiry date 08 October 2017; solvent DMSO
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 %; expiry date 12 October 2017; solvent DMSO
Details on test system and experimental conditions:
STUDY CONTROLS
- The solvent (vehicle) controls were performed in triplicate.
- The negative (untreated) controls were performed in triplicate to assess the spontaneous revertant colony rate.
- Sterility controls were performed for (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) the maximum dosing solution of the test item in the absence of S9-mix (singular test only).

MICROSOMAL ENZYME FRACTION
- The S9 microsomal fraction was pre-prepared using standardised in-house procedures.
- Lot number 05 June 2016 was used for the study.
- The certificate of S9 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 42228 09/16 and 42325 09/16).

BACTERIA
- Details of the five strains of bacteria used and their mutations are attached.
- All of the Salmonella strains were histidine dependent by virtue of a mutation through the histidine operon and were derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the “deep rough” (rfa) mutation they possessed a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through deletion of the uvrB bio gene, caused 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.
- In addition to a mutation in the tryptophan operon, the E. coli tester strain contained a uvrA DNA repair deficiency, which enhanced its sensitivity to some mutagenic compounds. This deficiency allowed 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.
- Bacteria used in the test were obtained from the University of California, Berkeley, on culture discs (04 August 1995) and the 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).
- 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 tires 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 fully miscible in sterile distilled water at 50 mg/mL. Sterile distilled water was therefore selected as the vehicle.
- The test item was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (41 %) of the test item.
- 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.

EXPERIMENT 1 – DOSE SELECTION
- Eight concentrations of the test item were assayed in triplicate against each tester strain (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate).
- The direct plate incorporation method was used.

EXPERIMENT 1 – WITHOUT METABOLIC ACTIVATION
- An aliquot (0.1 mL) of an 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 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).

EXPERIMENT 2 – DOSE SELECTION
- Based on the results of Experiment 1, the dose range chosen for Experiment 2 was 15 to 5000 µg/plate.
- Six test item dose levels were selected 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 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 due to the revertant colonies spreading slightly and distorting the actual plate count.

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.
- 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.
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.
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.
- 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 judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
- Statistical difference 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 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 (with and without metabolic activation) are given in Tables 2 and 3 for Experiment 1 (attached) and Tables 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 dose level of 5000 µg/plate.
- In the first mutation test, 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). 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 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 at any dose of test item, with and without metabolic activation (S9-mix).
- Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains at any dose level, with and without metabolic activation (S9-mix) in Experiment 2.
- The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range.
- All 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.

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 (30 May 2008) and the USA EPA OCSPP harmonised guideline – Bacterial Reverse Mutation Test.

 

METHODS

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 plus Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels. The investigation was performed in triplicate, with and without the addition of rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). 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 (sterile distilled water) 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, 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 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 the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial 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 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 item was considered to be non-mutagenic under the conditions of the test.