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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 August 2018 - 18 October 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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
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

Constituent 1
Chemical structure
Reference substance name:
1,2-dichloro-1,1,2-trifluoro-2-(trifluoromethoxy)ethane
EC Number:
219-094-1
EC Name:
1,2-dichloro-1,1,2-trifluoro-2-(trifluoromethoxy)ethane
Cas Number:
2356-53-8
Molecular formula:
C3Cl2F6O
IUPAC Name:
1,2-dichloro-1,1,2-trifluoro-2-(trifluoromethoxy)ethane
Test material form:
liquid
Details on test material:
Appearance: Colourless liquid
Storage conditions: At room temperature container flushed with nitrogen
Specific details on test material used for the study:
Because of the known volatility of the test substance, the study design was adapted and consisted in a pre-incubation at 20°C in closed vials before plating, in both assays.

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Dose-range finding test: (without and with S9; tester strains TA100 and WP2uvrA): 52, 164, 512, 1600 and 5000 µg/plate
First experiment: (without and with 5% (v/v) S9; tester strains TA1535, TA1537 and TA98): 52, 164, 512, 1600 and 5000 µg/plate
Second experiment: (without and with 10% (v/v) S9, tester strains WP2uvrA and TA100 TA1535, TA1537 and TA98): 492, 878, 1568, 2800, 5000 μg/plate
Vehicle / solvent:
- Vehicle used: Ethanol
- Justification for choice of vehicle: A previously performed solubility test showed that the test item could be dissolved in ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Remarks:
For details on positive control substances, see Table 1 and Table 2
Details on test system and experimental conditions:
Two individual experiments were performed. The dose range-finding study with tester strains TA100 and WP2uvrA was reported as part of the first experiment. Both experiments were pre-incubation assays. The first experiment was conducted with 5% (v/v) S9-mix. The second experiment was conducted with 10% (v/v) S9-mix

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 30 ± 2 minutes at 70 rpm at 20 ± 1°C
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

METHODS:
The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 20 ± 1°C in closed vials, 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 (1E9 cells/mL) of one of the tester strains, 0.05 mL of a dilution of the test item in a suitable solvent. 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°C for 48 ± 4 h.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.
- Other: precipitation of the test item was recorded

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 and evidence of test item precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9- mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Rationale for test conditions:
Due to volatility of the test substance, pre-incubation method was selected to favor contact with the substance.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle 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 the tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up 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.

Results and discussion

Test resultsopen allclose all
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:
not applicable
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:
not applicable
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
Untreated negative controls validity:
not applicable
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:
not applicable
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:
not applicable
Positive controls validity:
valid
Additional information on results:
PRECIPITATION
- No precipitation was observed

CYTOTOXICITY
no cytotoxicity was observed

OTHER
- There were no increases in the number of revertant colonies indicating a positive response for inducing mutagenicity.
- The negative historical control data ranges except the response for TA100 in the presence of S9-mix in the second experiment. However , the validity of the test was considered to be not affected since the mean number of revertant colonies showed a characteristic number of revertant colonies when compared against relevant historical control data.
- The strain-specific positive control values were within the laboratory historical control data ranges except the response for TA1537 in the presence of S9-mix in the second experiment, However, the value was still more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.
- In both the first and second assay, criteria for a negative response were met for all tester strains with and without metabolic activation.

Applicant's summary and conclusion

Conclusions:
Based on the results of an Ames test, performed according to EC guidelines; OECD guideline 471 and GLP principles, Methylic adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

An Ames test was performed according to EC guidelines; OECD guideline 471 and GLP principles with Methylic Adduct. Two valid pre incubation-assays were performed with tester strains WP2uvrA, TA100, TA1535, TA1537 and TA98. The first experiment was conducted with and without 5% (v/v) S9 -mix and the second experiment was conducted with and without 10% (v/v) S9 -mix. The tester strains showed negative responses up to and including 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No precipitation or cytotoxicity was observed. Not all acceptability criteria were met but since they had no effects on the results or validity of the study, the study was considered valid. Based on the results of this study Methylic Adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.