Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The Study Director signed the study plan on 27 Sep 2017. The experimental start date was 19 Feb 2018, and the experimental completion date was 12 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Appearance : clear colourless liquid
Test item storage : in the refrigerator (2-8°C)

Method

Target gene:
Histidine locus and tryptophan locus
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):
Source : Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535, TA1537, TA98, TA100; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA)]
Test concentrations with justification for top dose:
- 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 S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
- The highest concentration used in the subsequent mutation assays was 5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
- The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Vehicle / solvent:
dimethyl sulfoxide (Merck, Darmstadt, Germany)
Controls
Untreated negative controls:
yes
Remarks:
dimethyl sulfoxide (Merck, Darmstadt, Germany)
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
- Preparation of Test Item
No correction was made for the purity/composition of the test item. A solubility test was performed based on visual assessment. The test item formed a clear colorless solution in dimethyl sulfoxide (DMSO). The test item is extremely volatile. Therefore the time between preparation of the test item formulations and performance of the experiments was as short as possible. In the second experiment 2 separate formulations were prepared to keep the time in between as short as possible. Closed containers were used for the formulations. Test item concentrations were used within 2 hours after preparation in the dose range finding study, within 1 hour in the first mutation assay and within 20 minutes in the second mutation assay. Any residual volumes were discarded.

- 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 (109 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 35.8 - 38.5°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 Milli-Q water (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.

- First Experiment: Direct Plate Assay
In the main study, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. 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 (10E9 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO 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.

- Second Experiment: Pre-Incubation Assay
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 20 ± 1°C, 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), 0.1 mL of a fresh bacterial culture (10E9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO. The pre-incubation temperature deviated from the recommended range of 30 to 37°C (OECD 471). However since the boiling point of the test item was around 22°C, this was scientifically justified. After the pre-incubation period the solutions were added to 3 mL molten top agar. 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.


Rationale for test conditions:
See details on test system and conditions
Evaluation criteria:
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.

Critical computerized systems used in the study are listed below. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.
- REES Centron (SQL 2.0) : Temperature (laboratory facilities), Data collection
- Ames study Manager (v 1.23) Revertant Colony Count Collection

Results and discussion

Test resultsopen allclose all
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
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
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
Positive controls validity:
valid
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
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
Positive controls validity:
valid
Additional information on results:
- First Experiment: Direct Plate Assay
Test item was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.
Precipitation of test item on the plates was not observed at the start or at the end of the incubation period in any tester strain except in tester strains TA1535 (absence of S9-mix) and TA1537 (presence of S9-mix) where precipitate was observed at the highest dose level tested and in tester strain TA1535 in the presence of S9-mix where precipitate was observed at dose levels of 512 µg/plate and upwards.
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

- Second Experiment: Pre-Incubation Assay
To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of 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 the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitation of test item on the plates was not observed at the start or at the end of the incubation period.
There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

- conclusion :
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 independently repeated experiments.
The negative and 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.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay under the experimental conditions described in this report. 
Executive summary:

The objective of this study was to determine the potential of 1,1,1-trifluoroacetone (CAS RN 421-50-1) and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonellatyphimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichiacoli (E. coli) strain WP2uvrAin the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. The study procedures described in this report were based on the most recent OECD and EC guidelines. The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. 

In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. In all three experiments the test item did not precipitate on the plates except in the first mutation experiment in tester strains TA1535 (absence of S9-mix) and TA1537 (presence of S9-mix) where precipitate was observed at the highest dose level tested and in tester strain TA1535 in the presence of S9-mix where precipitate was observed at dose levels of 512 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. 

The negative and 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 test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay under the experimental conditions described in this report.