Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Off-white crystalline powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
E. coli WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Dose-range finding test: Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
First mutation experiment: 52, 164, 512, 1600 and 5000 μg/plate., based on the results of the dose-range finding test.
Second mutation experiment: 275, 492, 878, 1568, 2800 and 5000 μg/plate., based on the results of the dose-range finding test.
Vehicle / solvent:
The vehicle of the test item was dimethyl sulfoxide
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: Acridine Mutagen ICR 191
Remarks:
without metabolic activation
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, all strains, solvent: dimethyl sulfoxide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Cell Culture
Preparation of bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/ml). Freshly grown cultures of each strain were used for a test.
Agar plates: Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).
Top agar: Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.
Environmental conditions: All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.3 - 38.4 °C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic Activation System
S9-Fraction: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.
Preparation of S9-Mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 mL Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment..

TEST DESIGN

Dose-range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of CH02672 used in the subsequent mutation assays was 5000 μg/plate.

Direct Plate Assay
At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture
(109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item and either
0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Rationale for test conditions:
Guideline test conditions.
Evaluation criteria:
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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:
Dose-range Finding Test/First Mutation Experiment
Precipitation of CH02672 on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate. No precipitate was observed at the end of the incubation period.
Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in the tester strains TA1535, TA1537, TA98 and TA100 in the absence and presence of S9-mix.
No increase in the number of revertants was observed upon treatment with CH02672 under all conditions tested.

Second Mutation Experiment
To obtain more information about the possible mutagenicity of CH02672, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of
5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitation of CH02672 on the plates was observed at the start of the incubation period at concentrations of 2800 and 5000 μg/plate and no precipitate was observed at the end of the incubation period.
In the second mutation assay, cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA98 and TA100 in the absence and presence of S9-mix and in the tester strains TA1535 and TA1537 in the absence of S9-mix.
In the second mutation assay, no increase in the number of revertants was observed upon treatment with CH02672 under all conditions tested.

General discussion:
All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two experiments.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
The negative control values were within the laboratory historical control data ranges, except the response for WP2uvrA in the absence of S9-mix, first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (60 revertant colonies) when compared against relevant historical control data (59 revertant colonies), the validity of the test was considered to be not affected.

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this study it is concluded that CH02672 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The objective of this study was to determine the potential of CH02672 and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The study procedures described in this report were based on the most recent OECD 471 (1997) and EC 440 (2008) guidelines.

Batch CH02672/II/2 of CH02672 was an off-white crystalline powder. The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. CH02672 did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, a reduction of the bacterial background lawn and/or presence of microcolonies, was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 17 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. CH02672 did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 275 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strains TA1535, TA1537 and WP2uvrA in the presence of S9-mix and WP2uvrAin the absence of S9-mix.

Both in the absence and presence of S9-metabolic activation, CH02672 did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) nor in the number of revertant (Trp+) colonies in the tester strain WP2uvrA. These results were confirmed in a follow-up experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that CH02672 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.