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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 August 2013 - 19 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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: viscous
Details on test material:
Identification: NovaSpec 450Batch: TS8837Purity: 100% (UVCB)Expiry Date: Not suppliedStorage Conditions: Room temperature in the darkNo correction was made for purity.

Method

Species / strain
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):
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).Strains Genotype Type of mutations indicatedTA1537 his C 3076; rfa-; uvrB-: frame shift mutationsTA98 his D 3052; rfa-; uvrB-;R-factor : frame shift mutationsTA1535 his G 46; rfa-; uvrB-: base-pair substitutionsTA100 his G 46; rfa-; uvrB-;R-factor : base-pair substitutionsEscherichia coliStrain Genotype Type of mutations indicatedWP2uvrA trp-; uvrA-: base-pair substitution
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/B-Naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
The vehicle control used was tetrahydrofuran.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
Test Item
The test item was immiscible in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but was fully miscible in tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. Tetrahydrofuran was therefore selected as the vehicle.The test item was accurately weighed and approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer on the day of each experiment. Tetrahydrofuran is toxic to the bacterial cells at and above 50 µL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 µL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). 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. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicatepellets with a nominal pore diameter of 4 x 10-4 microns.Control ItemsVehicle and positive controls were used in parallel with the test item. The vehicle control used was tetrahydrofuran.
The positive control items used in the series of plates without S9-mix were as follows
:N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 µg/plate for WP2uvrA
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 3 µg/plate for TA100
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 5 µg/plate for TA1535
9-Aminoacridine (9AA): 80 µg/plate for TA1537
4-Nitroquinoline-1-oxide (4NQO): 0.2 uµg/plate for TA9

8In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolizing enzymes, were used in the series of plates with S9-mix at the following concentrations:
2-Aminoanthracene (2AA): 1 µg/plate for TA100
2-Aminoanthracene (2AA): 2 µg/plate for TA1535 and TA1537
2-Aminoanthracene (2AA): 10 µg/plate for WP2uvrA
Benzo(a)pyrene (BP): 5 µg/plate for TA98

Microsomal Enzyme Fraction
Lot No. PB/BNF S9 14 July 2013 was used in this study. The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/B-Naphthoflavone at80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed bycentrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 deg C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.The study was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedures) Act 1986. The conduct of the study may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process.S9-Mix and AgarThe S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL1.65 M KCl/0.4 M MgCl2 1.0 mL0.1 M Glucose-6-phosphate 2.5 mL0.1 M NADP 2.0 mL0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL Sterile distilled water14.5 mLA 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 2299373 09/17) 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 34989 09/13 and 35101 10/13).

Test Procedure
Test for Mutagenicity (Experiment 1 - Range-Finding Test) – Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). 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.
Without Metabolic Activation
0.025 mL of the appropriate concentration of test item or vehicle or 0.1 mL of appropriate positive control was added to 2 mL of trace amino-acid supplemented media (at approximately 45 deg C) 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. Each concentration of the test item, appropriate positive control, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the trace amino-acid supplemented media instead of phosphate buffer.All of the plates were incubated at 37 deg C± 3 deg 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).

Test for Mutagenicity (Experiment 2 - Main Test) – Pre-Incubation Method
As Experiment 1 (the range-finding test) was deemed negative, Experiment 2 (main test) was performed using the pre-incubation method in the presence andabsence of metabolic activation.
Dose selection
The dose range used for Experiment 2 (main test) was determined by the results of Experiment 1 (the range-finding test) and was 50 to 5000 µg/plate.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.025 mL of the test item formulation or solvent or 0.1 mL of appropriate positive control were incubated at37 deg C± 3 deg C for 20 minutes (with shaking) prior to addition of 2 mL of amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 deg C± 3 deg C for 20 minutes (with shaking) and addition of amino-acid supplemented media. All testing for this experiment was performed in triplicate.All of the plates were incubated at 37 deg C± 3 deg 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: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).All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the General Study Plan, Section 2.2.2 (negative controls). Combined historical negative and solvent control ranges for 2011 and 2012 are presented in Appendix 2.All tester strain cultures should be in the range of 0.9 to 9 x 109 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, both with or without metabolic activation. The historical ranges of the positive control reference items for 2011 and 2012 are presented within the report.There should be a minimum of four non-toxic test item dose levels. There should be no evidence of excessive contamination.
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. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).5. 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:
As above

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. 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.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 (range-finding test) and Table 4 and Table 5for Experiment 2 (main test) of the report. The results are also expressed graphically in Figure 1 to Figure 4 of the report. Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 2 of the report. A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 2.The maximum dose level of the test item in the range-finding test 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, in the first mutation test and consequently the same maximum dose level was used in the second 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, in the second mutation test. A test item precipitate (globular in appearance) was observed at 5000 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 in the range-finding test (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 in the main test (pre-incubation method). All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Tabulated data and figures are appended below.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negative NovaSpec 450 was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The vehicle (tetrahydrofuran) 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 range-finding test 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, in the first mutation test and consequently the same maximum dose level was used in the second 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, in the second mutation test. A test item precipitate (globular in appearance) was observed at 5000 ug/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 in the range-finding test (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 in the main test (pre-incubation method).

NovaSpec 450 was considered to be non-mutagenic under the conditions of this test.