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Toxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(3,5-dichloro-4-fluorophenyl)-2,2,2-trifluoroethanone
EC Number:
829-719-5
Cas Number:
1190865-44-1
Molecular formula:
C8H2Cl2F4O
IUPAC Name:
1-(3,5-dichloro-4-fluorophenyl)-2,2,2-trifluoroethanone
Test material form:
liquid

Method

Target gene:
The S. typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively. The S. typhimurium and E. coli strains are constructed to differentiate between base pair (TA1535, TA100, WP2 uvrA (pKM101), and WP2 (pKM101)) and frameshift (TA1537, TA98) mutations.
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Wistar rats (RjHan:WI; weight approx. 220 – 320 g, Janvier Labs, 53941 Saint-Berthevin Cedex, France) induced by peroral administration of 80 mg/kg b.w. phenobarbital (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) and by peroral administrations of ß-naphthoflavone (Acros Organics, 2440 Geel, Belgium) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene. The protein concentration in the S9 preparation was 30.3 mg/mL (lot no. 281119K, recertified 03 June 2020) in the pre-experiment / Experiment I and 33.0 mg/mL (lot no. 030920K) in Experiments Ia and II.
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment Ia: 1, 3, 10, 33, 100, 333 and 1000 µg/plate
Experiment II: 0.3, 1, 3, 10, 33, 100, 333 and 1000 µg/plate
Significant cytotoxicity was observed in experiment I. Furthermore, precipitation occurred at 2500 and 5000 µg/plate in the presence of S9 mix in experiment I.
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Remarks:
Sodium azide without S9 for TA1535 and TA100; 4-nitro-o-phenylene-diamine without S9 for TA1537, TA98; methyl methane sulfonate without S9 for WP2, 2-aminoanthracene with S9 for all strains
Details on test system and experimental conditions:
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µL bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL overlay agar

In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspensions were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
Evaluation criteria:
A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants of twice or above the spontaneous mutation rate of the corresponding solvent control is observed. A concentration dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration. An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
None

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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Since strong cytotoxic effects were observed in Experiment I eight concentrations were tested in Experiment II. 1000 µg/plate was chosen as the maximal concentration in Experiment II. The test item precipitated in the overlay agar in the test tubes in Experiment I at 5000 µg/plate in the presence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in Experiment I from 2500 to 5000 µg/plate in the presence of S9 mix. The undissolved particles had no influence on the data recording. Based on a lower top concentration of 1000 µg/plate no precipitation of the test substance occurred in the overlay agar in Experiments Ia and II neither in the test tubes nor on the incubated agar plates. No substantial increase in revertant colony numbers in any of the six tester strains was observed following treatment with the substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies consistent with the laboratory’s historical control data demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

Applicant's summary and conclusion

Conclusions:
The substance did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterial S. typhimurium and E. coli strains used and was found to be non-mutagenic.
Executive summary:

The potential of the substance to induce gene mutations in the plate incorporation test (Experiments I and Ia) and the pre-incubation test (Experiment II) using the Salmonella typhimurium (S. typhimurium) strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli (E. coli) strains WP2 uvrA (pKM101) and WP2 (pKM101) was studied under GLP to OECD TG 471. The concentrations were 3 to 5000 µg/plate in Experiment I, 1 to 1000 µg/plate in Experiment Ia and 0.3 to 1000 µg/plate in Experiment II. The plates incubated with the test item showed reduced background growth in all strains used with and without S9 mix. Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used with and without S9 mix. No relevant increase in revertant colony numbers of any of the six tester strains was observed following treatment with the substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls, which showed a distinct increase of induced revertant colonies consistent with the laboratory’s historical control data demonstrated the sensitivity of the test system and the efficacy of the S9 mix. Each batch of S9 was also tested with 2 pro-mutagens, benzo(a)pyrene and 2-aminoanthracene.

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