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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 November 2016 to 25 November 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:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
complementary test with TA1537 necessary with no impact on integrity or results of the experiment (see below)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
complementary test with TA1537 necessary with no impact on integrity or results of the experiment (see below)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
complementary test with TA1537 necessary with no impact on integrity or results of the experiment (see below)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
yes
Remarks:
complementary test with TA1537 necessary with no impact on integrity or results of the experiment (see below)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Appearance/physical state: Tan waxy solid
- 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:
post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.
Test concentrations with justification for top dose:
- Preliminary concentration range-finding test: 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate
- Initial Mutation Test, Confirmatory Mutation Test and Complementary Initial Mutation Test in Salmonella typhimurium TA1537 strain with and without metabolic activation: 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
Vehicle / solvent:
Acetone
Controlsopen allclose all
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 4-nitro-1,2-phenylenediamine
Remarks:
NPD (4 μg/plate for Salmonella TA98)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
SAZ (2 μg/plate for Salmonella TA100 and TA1535)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
9AA (50 μg/plate for Salmonella TA1537)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
MMS (2 μL/plate for E.coli WP2 uvrA)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
2AA (2 μg/plate for all Salmonella strains)
Positive control substance:
other: 2-aminoanthracene
Remarks:
2AA (50 μg/plate for E.coli WP2 uvrA)
Negative solvent / vehicle controls:
yes
Positive control substance:
other: Acetone
Negative solvent / vehicle controls:
yes
Positive control substance:
other: Dimethyl sulfoxide
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Positive control substance:
other: Distilled water
Details on test system and experimental conditions:
FORMULATION
- The appropriate vehicle and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were determined in a preliminary compatibility test.
- All dilutions in the main tests of test item were made in the testing laboratory using Acetone (approx. 12 minutes ultrasonic water bath).
- Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent.
- Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.
- No purity conversion was applied in the study.
- The respective test concentrations in the main tests are shown in Table 1 (below).

POSITIVE CONTROLS
- Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test.
- Positive control materials were selected based on the scientific literature, the experience of the Test Facility and the availability of historical control data.
- 2-aminoanthracene (2AA) Batch number of 10157819 was used in the Preliminary Concentration Range Finding Test and batch number of STBD3302V was used in the Main tests.

VEHICLE/SOLVENT CONTROLS
- Three vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive chemicals.

BACTERIAL STRAINS
- Source: All bacterial strains were received from MOLTOX (Molecular Toxicology Inc, Boone, North Carolina, USA) on 21 April 2015. True copies of original certificates plus other strain documentation were collected and stored in the Microbiological Laboratory of CiToxLAB Hungary Ltd.
- Genotypes: In addition to histidine or tryptophan mutation, each strain has additional mutations,
which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules.
- The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair. The genotypes of the tester strains used for mutagenicity testing are summarised in Table 3 (attached).
- Storage: The strains were stored at -80 ± 10ºC in the Culture Collection of the Microbiological Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- Confirmation of phenotype: The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames. Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of CiToxLAB Hungary Ltd.

SPONTANEOUS REVERSION OF TESTER STRAINS
- Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (in Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
- Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011 to 2014 were (as guide) as follows: Salmonella typhimurium TA98: 9-46, TA100: 54-210, TA1535: 1-46, TA1537: 1-24, Escherichia coli WP2 uvrA: 11-82. More detailed historical
control data are shown in Appendix 7 (attached).

PROCEDURE FOR GROWING CULTURES
- The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay.
- The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory Water Bath Shaker.

VIABILITY OF THE TESTING CULTURES
- The viability of each testing culture was determined by plating 0.1 mL of the 10E05, 10E06, 10E07 and 10E08 dilutions prepared by sterile physiological saline on nutrient agar plates.
- The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

MEDIA
- The supplier, batch number and expiry date of chemicals used in the investigation is summarised in Table 5 (attached).
- Minimal glucose agar typically contained glucose (20.0 g/L); magnesium sulfate (0.2 g/L); citric acid (2.0 g/L); dipotassium hydrogenphosphate (10.0 g/L); sodium ammonium hydrogenphosphate (3.5 g/L); agar (13.0 g/L); distilled water of a quantity sufficient to give a volume of 1000 mL.
- Minimal glucose agar plates were provided by Merck (Batch number: 140464, Expiry date: 10 January 2017 was used in the Preliminary Concentration Range Finding Test; Batch number: 141537, Expiry date: 21 March 2017 was used in the Initial Mutation Test, in the Complementary Initial Mutation Test and Confirmatory Mutation Test). Certificate of Analysis was obtained from the Supplier.
- Nutrient Broth No 2 (25.0 g made up to a volume of 1000 mL with distilled water) was sterilised in an autoclave at 121 °C.
- Nutrient agar (20.0 g made up to a volume of 1000 mL with distilled water) was sterilised in an autoclave at 121 °C.
- Top agar for Salmonella typhimurium strains contained agar bacteriological (4.0 g); NaCl (5.0 g); sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed at 121 °C in an autoclave.
- Histidine-biotin solution (0.5 mM) contained D-biotin (FW 244.31) 122.2 mg; L-histidine HCl:H2O (FW 209.63) 104.8 mg; sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed by filtration using a 0.22 µm membrane filter.
- Complete top agar for Salmonella typhimurium strains contained 0.5 mM histidine-biotin solution (100 mL) and agar solution (900 mL).
- Trptophan solution (2 mg/mL) contained L-tryptophan (FW 204.23) 2000 mg and sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed by filtration using a 0.22 µm membrane filter.
- Complete top agar for the Escherichia coli strain contained Nutrient Broth No 2 (50 mL), 2 mg/mL tryptophan solution (2.5 mL) and agar solution 947.5 mL.

METABOLIC ACTIVATION SYSTEM
- Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.
- The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of CiToxLAB Hungary Ltd. according to Ames et al. and Maron and Ames.
- The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
- The supplier, batch number and expiry date of chemicals used are summarised in Table 5 (attached).

INDUCTION OF LIVER ENZYMES
- Male Wistar rats (345-441 g, animals were 9 weeks old at the initiation) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days.
- Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed.
- Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels.
- Initiation of the induction of liver enzymes used for preparation S9 used in this study was 25 July 2016.

PREPARATION OF RAT LIVER HOMOGENATE S9 FRACTION
- On Day 4, the rats were euthanised and the livers were removed aseptically using sterile surgical tools.
- After excision, livers were weighed and washed several times in 0.15 M KCl.
- The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenised.
- Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained.
- The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10 °C.
- The date of preparation of S9 fraction for this study was 28 July 2016 (CiToxLAB code: E12440).
- The sterility of the preparation was confirmed. The protein concentration of the preparation was determined by a chemical analyser at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd.
- The mean protein concentration of the S9 fraction used was determined to be 29.2 g/L.
- The biological activity in the Salmonella assay of S9 was characterized using the two mutagens (2-Aminoanthracene and Benzo(a)pyrene) that requires metabolic activation by microsomal enzymes.
- The batch of S9 used in this study functioned appropriately.

S9 MIX
- Salt solution for the S9 mix contained NADP Na (7.66 g); D-glucose-6-phosphate Na (3.53 g); MgCl2.6H20 (4.07 g); KCl (6.15 g); sufficient distilled water to give 1000 mL. Sterilisation was performed by filtration through a 0.22 µm membrane filter.
- The complete S9 mix was freshly prepared and contained ice cold 0.2 M sodium phosphate buffer pH 7.4 (500 mL); S9 rat liver homogenate (100 mL); salt solution for S9 mix (400 mL).
- The S9 mix was kept in an ice bath prior to addition to the culture medium.
- Sodium phosphate buffer 0.2 M (pH 7.4) solution A contained Na2HPO4 (71.63 g) and sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed in an autoclave at 121 °C.
- Sodium phosphate buffer 0.2 M (pH 7.4) solution B contained Na2PO4.H2O (27.6 g) and sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed in an autoclave at 121 °C.
- Sodium phosphate buffer pH 7.4 contained solution A (880 mL) and solution B (120 mL).

TEST PROCEDURE
- The study included a preliminary compatibility test, a preliminary range finding test (informatory toxicity test), an initial mutation test, a confirmatory mutation test and a complementary confirmatory mutation test.
- The plate incorporation method was used.

CONCENTRATIONS
- Concentrations were selected on the basis of the preliminary compatibility test and preliminary range finding test (informatory toxicity test).
- The same concentrations were used in the Initial Mutation Test, Complementary Initial Mutation Test and Confirmatory Mutation Test.

PRELIMINARY COMPATIBILITY TEST
- Solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF) and Acetone. The test item was insoluble in Distilled water and in DMSO (after 2-minutes incubation ultrasonic water bath) at 100, 50 and 33.3 mg/mL concentration. Partial dissolution was observed at 100, 50 and 33.3 mg/mL (precipitate was observed) concentration using DMF (after 2-minutes
incubation ultrasonic water bath). Partial dissolution was observed at 100, 50 and 33.3 mg/mL (quickly settling formulation) concentration using Acetone and the formulation at 33.3 mg/mL concentration using Acetone as vehicle (after 2-minutes incubation in an ultrasonic water bath) was a homogeneous formulation.
- Note: The test item was not a homogeneous formulation at 100 and 50 mg/mL concentration after 2-
minutes incubation ultrasonic water bath.
- Based on the results, Acetone was selected as vehicle (solvent) for the study. The obtained stock formulation (150 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility (Preliminary Compatibility Test).

PRELIMINARY CONCENTRATION RANGE-FINDING TEST (INFORMATORY TOXICITY TEST)
- Based on the available information and the solubility and compatibility test, 33.33 mg/mL stock solution was prepared in Acetone, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

TEST ITEM CONCENTRATIONS IN THE MUTAGENICITY TESTS
- Based on the results of the preliminary test, 33.33 mg/mL stock solution was prepared in Acetone, which was diluted by serial dilutions in six steps to obtain seven dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
- Examined concentrations in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Initial Mutation Test in Salmonella typhimurium TA1537 strain with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5μg/plate.

CONTROL GROUPS USED IN THE TESTS
- Strain-specific positive and negative (vehicle/solvent) controls, both with and without metabolic activation were included in each test.
- In addition, untreated control was used demonstrating that the chosen vehicle (solvent) induced no deleterious or mutagenic effects.
- The control groups are summarised in Table 4 (below).
- If the solvent of the positive control substance differed from the vehicle (solvent) of the test item, both solvents were run in the assay.

EXPOSURE PROCEDURE
- A standard plate incorporation procedure was performed as an Initial Mutation Test, Complementary Initial Mutation Test and Confirmatory Mutation Test. Bacteria (cultured in Nutrient Broth No.2) were exposed to the
test item both in the presence and absence of an appropriate metabolic activation system.
- Note: The Confirmatory Mutation Test was performed using the plate incorporation method because the required treatment volume (150 μL) of Acetone as vehicle is more suitable for this method than the pre-incubation method.
- Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
- Tubes contained top agar (2000 µL); vehicle (solvent) or test item solution (or reference controls) 150 (50 µL); overnight culture of test strain (100 µL); phosphate buffer (pH 7.4) or S9 mix (500 µL).
- Note: Treatment volume was 150 μL for test item formulation and its vehicle control in the main experiments; treatment volume was 50 μL for positive control substance formulations and their vehicle controls.
- This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls.
- After preparation, the plates were incubated at 37 °C for 48 hours.

VALIDITY CRITERIA
- The number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests.
- At least five analysable concentrations were presented in all strains of the main tests.

CRITERIA FOR A POSITIVE RESPONSE
- A dose–related increase in the number of revertants occurred and/or a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
- An increase was considered biologically relevant if:
(i) The number of reversions was more than two times higher than the reversion rate of the
negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains.
(ii) The number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
- According to the guidelines, statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

CRITERIA FOR A NEGATIVE RESPONSE
- A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Evaluation criteria:
EVALUATION OF EXPERIMENTAL DATA
- Colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard
deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
- The Mutation factor (MF) = mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Results and discussion

Test resultsopen allclose all
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:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY COMPATIBILITY TEST
- Based on the available information and the results of the solubility testing, Acetone was selected as vehicle (solvent) of the study.
- The results of the Preliminary Compatibility Test are summarised in Table 6 (attached).

PRELIMINARY CONCENTRATION RANGE-FINDING TEST
- In the Preliminary Concentration Range Finding Test (Informatory Toxicity Test), the plate incorporation method was used. This test was performed using Salmonella typhimurium TA98 and TA100 strains in the presence and absence of metabolic activation system (± S9 mix) with appropriate untreated, negative (vehicle/solvent) and
positive controls. In the test, each sample (including the controls) was tested in triplicate.
- Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test.
- In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
- Precipitate/slight precipitate was observed in the both tester strains with and without metabolic activation at the concentrations of 5000, 2500 and 1000μg/plate.
- No inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding Test.
- The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 and in Appendix 3 (attached).

INITIAL AND CONFIRMATORY MUTATION TESTS
- In the Initial Mutation Test, Confirmatory Mutation Test and Complementary Mutation Test, the plate incorporation method was used.
- The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain in the presence and absence of a metabolic activation system (± S9 mix) with appropriate untreated, negative (vehicle/solvent) and
positive controls.
- The Complementary Initial Mutation Test was carried out using Salmonella typhimurium TA1537 strain in the presence and absence of a metabolic activation system (-S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests, each sample (including the controls) was tested in triplicate.
- Note: In the Initial Mutation Test in Salmonella typhimurium TA1537 strain was infected. Therefore, an additional experiment (Complementary Initial Mutation Test) was performed in this strain in an additional experimental period (Experimental Period III) to complete the data. The experimental conditions were the same as in the Confirmatory Mutation Test. Results of the invalid experiment were not reported; however, all data are kept and archived in the raw data binder.
- Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Initial Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
- In the Initial Mutation Test and in the Complementary Initial Mutation Test (using the plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 50μg/plate concentration with metabolic activation (the observed mutation factor value was 1.50). However, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- In the Confirmatory Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA98 at 15.81μg/plate concentration without metabolic activation (the observed mutation factor value was 1.30). However, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
- Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological
variability of the test system.
- Precipitate/slight precipitate was detected on the plates in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Initial Mutation Test in all examined strains with and without metabolic activation at 5000, 1581 and 500 μg/plate concentrations.
- Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Mutation Test.
- The experimental results (revertant colony numbers per plate, mutation factors, and standard deviations) are summarized in Tables 8-9 and Appendices 4-6 (attached)

VALIDITY OF THE TESTS
- Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.
- The examined concentration range was considered to be adequate as concentrations up to maximum recommended concentration (5000 μg/plate) were examined in the main tests. At least five analysable concentrations were presented in all strains with and without metabolic activation.
- The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Applicant's summary and conclusion

Conclusions:
The experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item demonstrated no mutagenic activity in the bacterial tester strains used in the study.
Executive summary:

GUIDELINE

The test item was investigated for potential mutagenic activity using the bacterial reverse mutation assay. The study was conducted in accordance with the Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No 471, "Bacterial Reverse Mutation Test" (21 July 1997), EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test", EPA 712-C-98-247 (August 1998) and Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse Mutation Test Using Bacteria" (30 May 2008). The method also conformed to conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

 

METHODS

Experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Plate Incorporation Method) and a Complementary Mutation Test (Plate Incorporation Method).

 

Based on the results of a solubility tests, the test item was formulated in Acetone. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Initial Mutation Test in Salmonella typhimurium TA1537 strain with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5μg/plate.

 

RESULTS

In the Initial Mutation Test, Confirmatory Mutation Tests and the Complementary Initial Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

 

Inhibitory, cytotoxic effects of the test item were not detected in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation.

 

Precipitate/slight precipitate was detected on the plates in the Preliminary Concentration Range Finding Test, in the Initial Mutation Test, Confirmatory Mutation Test in all examined bacterial strains with and without metabolic activation at several concentrations.

 

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

 

CONCLUSION

The experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item demonstrated no mutagenic activity in the bacterial tester strains used in the study.