Registration Dossier
Registration Dossier
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EC number: 911-467-3 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate was tested in vitro for genetic mutations induction the bacterial reverse mutation assay and in mammalian cells and for clastogenic effects in a chromosomal aberration test. Based on the results it is concluded that the Reaction Mass of TFSK/TFAK does not exhibit any potential for mutagenicity.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 04 Nov 2011 to 10 may 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP Study, OECD 471 compliant
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 2010-12-16
- Type of assay:
- bacterial reverse mutation assay
- 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:
- Cofactor-supplemented post-mitochondrial fraction (S9 fraction) prepared by CiToxLAB and obtained from the liver of male Wistar rats treated with with phenobarbital and B-naphthoflavone at 80 mg/kg/day by oral gavage for three consecutive days.
- Test concentrations with justification for top dose:
- 5000; 1581, 500; 158.1, 50; 15.81 and 5 μg active ingredients/plate.
A preliminary solubility test was performed. The solubility of the test item was examined in Distilled water, Dimethyl sulfoxide (DMSO) and N,N-Dimethylformamide (DMF). The test item was soluble in Distilled water at 100 mg/mL concentration; while the use of DMSO or DMF resulted an opalescent formulation at the same concentration. Based on these solubility results, Distilled water was selected for solvent of the study. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Distilled Water
- Justification for choice of solvent/vehicle: The test item was soluble in Distilled water at 100 mg/mL concentration; while the use of DMSO or DMF resulted an opalescent formulation at the same concentration. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: see details on test system and conditions
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); with or without preincubation (for initial mutation test and confirmatory mutation test respectively)
POSITIVE CONTROLS :
-Without activation :
-Salmonella TA98 : 4 µg/plate of 4-nitro-1,2-phenylene-diamine (NPD) in DMSO
-Salmonella TA100 and TA1535 : 2 µg/plate of Sodium azide (SAZ) in Distilled water
-Salmonella TA1537 : 50 µg/plate of 9-aminoacridine (9AA) in DMSO
-E.coli WP2 uvrA : 2 µL/plate of Methyl-methanesulfonate (MMS) in Distilled water
-With activation :
-all Salmonella strains : 2 µg/plate of 2-aminoanthracene (2AA) in DMSO
-E.coli WP2 uvrA : 50 µg/plate of 2-aminoanthracene (2AA) in DMSO
DURATION
- Preincubation period: none for the initial mutation test. For confirmatory mutation test, the test item solution (or solvent or reference control) (50 µL), the bacterial culture (100 µL) and the S9 mix or phosphate buffer (500 µL) were incubated for 20 min at 37ºC in a shaking incubator before the overlaying
- Exposure duration: 48 hours
- Expression time (cells in growth medium): the number of revertants is determined at the end of the exposure time.
NUMBER OF REPLICATIONS: 3
NUMBER OF CELLS EVALUATED: all revertants per plates are counted
OTHER EXAMINATIONS:
- Other: Visual examination of the plates was performed : precipitation or signs of growth inhibition were reported
The plate incorporation method was performed as follow : the test item solution (or solvent or reference control) (50 µL), the bacterial culture (100 µL) and the S9 mix or phosphate buffer (500 µL) and the molten top agar (2000 µL) were mixed in individual tubes at 45°C and poured on the surface of minimal agar plates. - Evaluation criteria:
- Criteria for Validity:
The study was considered valid if:
-The number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
-at least five analyzable concentrations were presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
-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:
-in Salmonella typhimurium TA100 strain the number of reversion is more than two times higher than the spontaneous reversion rate of the solvent control plates;
-in Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA strains the number of reversions is more than three times higher than the spontaneous reversion rate of the solvent control plates.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produces 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. - Statistics:
- No statistical analysis was performed
- 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:
- 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
- Untreated negative controls validity:
- valid
- 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:
- 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
- Untreated negative 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
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: A Preliminary Range Finding Test was performed with the plate incorporation method. The test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. The concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
The observed numbers of revertant colonies were mostly in the normal range. Minor difference (slightly higher number of revertant colonies compared to the solvent control plates) was observed in one case. However, it had no biological significance and was considered as reflecting the variability of the test system.
No insolubility or signs of cytotoxicity were observed in the preliminary experiment.
COMPARISON WITH HISTORICAL CONTROL DATA:
Untreated, negative (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. At least five analyzable concentrations were presented in all strains of the main tests.
The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.
ADDITIONAL INFORMATION ON CYTOTOXICITY: Slight inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1537 bacterial strain at the highest examined concentration (5000 μg active ingredients/plate ) with and without metabolic concentration. - Conclusions:
- The Reaction mass of TFSK/TFAK had no mutagenic activity in the tested strains with or without activation up to 5000 µg (active ingredient)/plate.
- Executive summary:
The test item Reaction mass of TFSK/TFAKwas tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The 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 coliWP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/b-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.
The examined test item concentrations in the Initial Mutation Test and Confirmatory Mutation Test were: 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg active ingredients/plate.
In the Initial Mutation Test and Confirmatory Mutation Tests none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect, both with and without activation, in any of the five strains. The reported data of this mutagenicity assay show that under 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. In conclusion, the test item Reaction mass of TFSK/TFAK did not show mutagenic activity this study.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- between 2011-10-21 to 2012-06-19
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study, OECD 473 compliant
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 2010-12-16
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 5000; 2500; 1250; 625; 312.5; 156.25 and 78.125 µg active ingredients/mL
- Vehicle / solvent:
- - Vehicle: Dissolved water
- Justification for choice of solvent/vehicle: The test item was found to be soluble in Distilled water at 500 mg/mL concentration. - Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- POSITIVE CONTROLS :
Without S9 mix
- Ethyl methanesulfonate dissolved in DMEM at a final concentration of 0.4 μL/mL (28-hour harvesting time) or 1.0 μL/mL (20-hour harvesting time).
With S9 mix
- Cyclophosphamide dissolved in sterile physiological saline solution (0.9% NaCl infusion) at a final concentration of 6.0 μg/mL.
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: yes approximately 24 hours
- Exposure duration:
+ first assay: 3 hours with or without S9-mix (harvest 20 hours from the beginning of treatment)
+ second assay: 3 hours with S9 mix and 20 hours without S9 mix (harvest 28 hours from the beginning of treatment)
- Expression time (cells in growth medium): Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 105 cells/dish concentration
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: At least one hundred metaphases* with 22 +/- 2 chromosomes (dicentric chromosomes were counted as two chromosomes)
*Note: The examination of slides from a culture was halted when 15 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
OTHER EXAMINATIONS:
- Determination of polyploidy: yes (Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc). )
- Determination of endoreplication: yes (Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids. Marked reductions in the numbers of cells on the slides were recorded if needed.)
- Other: The Vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture - Evaluation criteria:
- The assay was considered valid, if the following criteria were met:
- The solvent control data were within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induced increases in the aberration frequency, which were significant.
The test item was considered to have shown clastogenic activity in this study if all of the following criteria were met:
- Increases in the frequency of metaphases with aberrant chromosomes were observed at one or more test concentrations (only data without gaps will be considered).
- The increases were reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases were statistically significant.
- The increases were not associated with large changes in pH or osmolarity of the treated cultures.
The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship was considered to support the conclusion.
The test item was concluded to have given a negative response if no reproducible, statistically significant increases were observed. - Statistics:
- For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Water solubility: 500 mg/mL
- Precipitation: No data
RANGE-FINDING/SCREENING STUDIES: No
COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous aberration frequencies of the solvent controls in the performed experiments were within the historical control range of the testing laboratory.
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the both assay, no cytotoxicity were observed in the final treatment medium at the end of the treatment either in the presence or absence of metabolic activation system. - Conclusions:
- Negative with and without metabolic activation
No cytotoxicity was observed up to 5000 µg of active ingredient/mL - Executive summary:
The Reaction mass of TFSK/TFAK was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells according to OECD 473 and under GLP. The test item was the material as supplied, but the dose concentrations reported were adjusted for the μg active ingredients/mL concentration (based on a purity figure of 36.5% supplied by the Sponsor). The material supplied was dissolved in Distilled water and was examined up to the highest recommended concentration of 5000 μg/mL (as μg active ingredients/mL concentration).
In the performed independent Chromosome Aberration Assays using duplicate cultures, at least 200 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each test item treated, negative (solvent) and positive control sample.
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 5000; 2500; 1250; 625; 312.5; 156.25 and 78.125 μg active ingredients/mL.
In Assay 1, no cytotoxicity and no insolubility were observed either in the presence or absence of the metabolic activation system. No large changes were detected in pH or osmolality at the evaluated concentrations. Therefore, concentrations of 5000; 2500 and 1250 μg active ingredients/mL (a total of three) were chosen for evaluation in both cases based on the observed results of the visual examination.
None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations either in the absence or in the presence of metabolic activation. This assay was considered as negative.
In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 5000; 2500; 1250; 625; 312.5; 156.25 and 78.125 μg active ingredients/mL.
In Assay 2, similarly to the first experiment, no cytotoxicity and insolubility were observed in the final treatment medium at the end of the treatment either in the presence or absence of metabolic activation system. No large changes were detected in pH or osmolality at the evaluated concentrations. Therefore, concentrations of 5000; 2500 and 1250 μg active ingredients/mL (a total of three) were chosen for evaluation in both cases based on the observed results.
None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations either in the absence or in the presence of metabolic activation. This assay was also considered as negative, thus confirmed the results of the first test.
The occurrence of polyploid and endoreduplicated metaphases was detected in the main tests. A polyploid metaphase was found in some cases in the negative (solvent) control, positive control or test item treated samples in the performed experiments. No endoreduplicated metaphases were found in the samples of the performed experiments except for one case (one endoreduplicated metaphase was detected in the samples of 5000 μg active ingredients/mL in Assay 1 experiment without metabolic activation).
These results indicated that the test item did not have potential to induce numerical chromosome aberrations or inhibit cell cycle progression under the conditions of this study.
The negative (solvent) control data were within the laboratory’s normal range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The tests were considered to be valid.
In conclusion, no induction of chromosome aberrations in Chinese hamster V79 cells was observed following treatment with Reaction mass of TFSK/TFAK up to 5000 μg active ingredients/mL concentration; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study. Therefore, Reaction mass of TFSK/TFAK is considered non-clastogenic in this test system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 27 Sep 2018 to 23 Jul 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Also according to GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- 2016
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 11 Feb 2019
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Thymidine Kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: TK +/- 3.7.2C from ATCC CRL-9518
For cell lines:
- Absence of Mycoplasma contamination: confirmed
- Number of passages if applicable: 9
- Periodically ‘cleansed’ of spontaneous mutants: yes]
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: cells were maintained at 35.8-37.5 °C in a humified air with 5.0% CO2. Cell medium was RPMI 1640 and RPMI 0-20 was prepared by adding horse serum. RPMI 10 was used during cell culture and treatment, RPMI 0 was used during cell treatement, RPMI 20 was used during cell cloning culture. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : in-house, prepared from male SD rats induced with Aroclor 1254
- concentration or volume of S9 mix and S9 in the final culture medium : 10% S9 in S9 mix
- quality controls of S9: before use S9 is tested for its sterility, protein content and capacity to activate known mutagens. - Test concentrations with justification for top dose:
- 8.192, 20.48, 51.2, 128, 320, 800 and 2000 µg/ml
2000 µg/ml is the highest concentration recommended in the OECD TG 490 and as preliminary tests did not show any precipitation or cytotoxicty, it was used as the highest dose. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: RPMI 10%
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 3x10^5
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3h with and without S9, 24h without S9
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 12d the for 3h treatment group and 11d for the 24h treatment group
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used: trifluorothymidine, 3 µg/ml, 12d the for 3h treatment group and 11d for the 24h treatment group
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2000 cells per well
- Criteria for small (slow growing) and large (fast growing) colonies: large colony = size the diamter not less than a quarter of diameter of the well, morphology thin and dispersive; small colony = diameter less than a quater of diameter of the well, morphology compact and nublly
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG) - Evaluation criteria:
- When IMF(s) at one or several dose levels are more than the GEF of 126 x 10^-6, and the increase is concentration-related and/or replicated, the result is evaluated as positive;
The result is evaluated as negative if none of the above critera is met. - Species / strain:
- mouse lymphoma L5178Y cells
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitation observed
STUDY RESULTS
- Concurrent vehicle negative and positive control data
Criteria for the solvent and positive controls were all met. The test is validated.
Detailed results are provided in Table 1 see 'Any other information on results'. - Conclusions:
- The results of this study indicate that the test item is non-mutagenic to the cultured L5178Y mouse lymphoma cells under the conditions of this study.
- Executive summary:
The test item has been tested for mutagenicity in mouse lymphoma L5178Y cells according to OECD TG 490, and in compliance with GLP. No test-substance induced increase in the number of mutations was observed when tested up to limit concentration in the presence and absence of metabolic activation. The result of the first experiment, 3-hour exposure, with and without metabolic activation, was confirmed in a second experiment, 24-hour exposure without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.
Referenceopen allclose all
Summary Table of the Initial Mutation Test
Concentrations
|
Mean |
Salmonella typhimuriumtester strains |
Escherichia coli |
|||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||
Untreated control |
Mean |
25.3 |
35.3 |
85.7 |
109.7 |
8.0 |
9.3 |
11.0 |
9.0 |
26.3 |
37.3 |
|
MF |
1.17 |
1.16 |
1.00 |
0.99 |
1.50 |
1.22 |
1.74 |
1.17 |
0.71 |
0.80 |
||
DMSO |
Mean |
24.7 |
35.7 |
-- |
112.3 |
-- |
8.3 |
12.3 |
10.7 |
-- |
41.7 |
|
MF |
1.14 |
1.18 |
-- |
1.02 |
-- |
1.09 |
1.95 |
1.39 |
-- |
0.89 |
||
Distilled water control |
Mean |
21.7 |
30.3 |
86.0 |
110.3 |
5.3 |
7.7 |
6.3 |
7.7 |
37.3 |
46.7 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
||
5000 |
Mean |
24.7 |
33.3 |
81.3 |
89.3 |
7.3 |
9.7 |
5.7 |
9.7 |
31.7 |
37.3 |
|
MF |
1.14 |
1.10 |
0.95 |
0.81 |
1.38 |
1.26 |
0.89 |
1.26 |
0.85 |
0.80 |
||
1581 |
Mean |
25.3 |
34.3 |
89.0 |
113.0 |
9.3 |
11.7 |
6.7 |
11.7 |
30.0 |
38.0 |
|
MF |
1.17 |
1.13 |
1.03 |
1.02 |
1.75 |
1.52 |
1.05 |
1.52 |
0.80 |
0.81 |
||
500 |
Mean |
26.0 |
32.0 |
82.0 |
114.3 |
6.3 |
8.7 |
7.3 |
9.3 |
24.7 |
28.7 |
|
MF |
1.20 |
1.05 |
0.95 |
1.04 |
1.19 |
1.13 |
1.16 |
1.22 |
0.66 |
0.61 |
||
158.1 |
Mean |
24.7 |
40.0 |
85.0 |
105.0 |
8.3 |
12.7 |
6.3 |
9.7 |
24.3 |
41.3 |
|
MF |
1.14 |
1.32 |
0.99 |
0.95 |
1.56 |
1.65 |
1.00 |
1.26 |
0.65 |
0.89 |
||
50 |
Mean |
25.3 |
37.3 |
80.3 |
107.7 |
10.3 |
11.0 |
8.0 |
7.0 |
25.0 |
33.7 |
|
MF |
1.17 |
1.23 |
0.93 |
0.98 |
1.94 |
1.43 |
1.26 |
0.91 |
0.67 |
0.72 |
||
15.81 |
Mean |
29.7 |
37.7 |
81.3 |
99.0 |
9.7 |
10.7 |
10.3 |
7.3 |
28.0 |
35.7 |
|
MF |
1.37 |
1.24 |
0.95 |
0.90 |
1.81 |
1.39 |
1.63 |
0.96 |
0.75 |
0.76 |
||
5 |
Mean |
21.0 |
32.7 |
77.7 |
118.0 |
8.3 |
11.3 |
6.3 |
10.0 |
25.7 |
38.3 |
|
MF |
0.97 |
1.08 |
0.90 |
1.07 |
1.56 |
1.48 |
1.00 |
1.30 |
0.69 |
0.82 |
||
NPD (4mg) |
Mean |
200.0 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
MF |
8.11 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2172.0 |
-- |
2186.7 |
-- |
202.7 |
-- |
222.0 |
-- |
-- |
|
MF |
-- |
60.90 |
-- |
19.47 |
-- |
24.32 |
-- |
20.81 |
-- |
-- |
||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
208.0 |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
4.99 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1452.0 |
-- |
1465.3 |
-- |
-- |
-- |
-- |
-- |
|
MF |
-- |
-- |
16.88 |
-- |
274.75 |
-- |
-- |
-- |
-- |
-- |
||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
308.0 |
-- |
-- |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
24.97 |
-- |
-- |
-- |
||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1236.7 |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
33.13 |
-- |
||
Summary table ofChromosome Aberration Assay 1
Concentration (μg/mL) |
Time of Treatment / Sampling |
Relative Survival# |
Insolubility## |
Mean % aberrant cells### |
|
Reaction mass of TFSK/TFAK without metabolic activation (-S9) |
|||||
Negative (solvent) control |
3h/ 20h |
100 |
– |
1.0 |
|
5000μg active ingredients/mL |
3h/ 20h |
85 |
– |
2.0 |
|
2500μg active ingredients/mL |
3h/ 20h |
91 |
– |
0.5 |
|
1250μg active ingredients/mL |
3h/ 20h |
77 |
– |
1.0 |
|
625μg active ingredients/mL |
3h/ 20h |
90 |
– |
NE |
|
312.5μg active ingredients/mL |
3h/ 20h |
88 |
– |
NE |
|
156.25μg active ingredients/mL |
3h/ 20h |
100 |
– |
NE |
|
78.125μg active ingredients/mL |
3h/ 20h |
84 |
– |
NE |
|
Positive control |
3h/ 20h |
71 |
– |
12.5*** |
|
Reaction mass of TFSK/TFAK with metabolic activation (+S9) |
|||||
Negative (solvent) control |
3h / 20h |
100 |
– |
1.0 |
|
5000μg active ingredients/mL |
3h / 20h |
116 |
– |
1.0 |
|
2500μg active ingredients/mL |
3h / 20h |
120 |
– |
1.5 |
|
1250μg active ingredients/mL |
3h / 20h |
94 |
– |
2.5 |
|
625μg active ingredients/mL |
3h / 20h |
94 |
– |
NE |
|
312.5μg active ingredients/mL |
3h / 20h |
88 |
– |
NE |
|
156.25μg active ingredients/mL |
3h / 20h |
90 |
– |
NE |
|
78.125μg active ingredients/mL |
3h / 20h |
105 |
– |
NE |
|
Positive control |
3h / 20h |
61 |
– |
90.9*** |
|
Negative (solvent) control: 2 (v/v) % Distilled water
Positive control (-S9): Ethyl methanesulfonate, 1.0 µL/mL
Positive control (+S9): Cyclophosphamide, 6.0 µg/mL
NE: not evaluated
#: compared to the negative (solvent) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
Summary table ofChromosome Aberration Assay 2
Concentration (μg/mL) |
Time of Treatment / Sampling |
Relative Survival# |
Insolubility## |
Mean % aberrant cells### |
|
Reaction mass of TFSK/TFAK without metabolic activation (-S9) |
|||||
Negative (solvent) control |
20h/ 28h |
100 |
– |
0.5 |
|
5000μg active ingredients/mL |
20h / 28h |
76 |
– |
0.5 |
|
2500μg active ingredients/mL |
20h / 28h |
85 |
– |
1.0 |
|
1250μg active ingredients/mL |
20h / 28h |
99 |
– |
0.0 |
|
625μg active ingredients/mL |
20h / 28h |
100 |
– |
NE |
|
312.5μg active ingredients/mL |
20h / 28h |
113 |
– |
NE |
|
156.25μg active ingredients/mL |
20h / 28h |
104 |
– |
NE |
|
78.125μg active ingredients/mL |
20h / 28h |
110 |
– |
NE |
|
Positive control |
20h/ 28h |
69 |
– |
42.3*** |
|
Reaction mass of TFSK/TFAK with metabolic activation (+S9) |
|||||
Negative (solvent) control |
3h / 28h |
100 |
– |
0.0 |
|
5000μg active ingredients/mL |
3h / 28h |
109 |
– |
1.0 |
|
2500μg active ingredients/mL |
3h / 28h |
108 |
– |
0.0 |
|
1250μg active ingredients/mL |
3h / 28h |
117 |
– |
1.5 |
|
625μg active ingredients/mL |
3h / 28h |
107 |
– |
NE |
|
312.5μg active ingredients/mL |
3h / 28h |
94 |
– |
NE |
|
156.25μg active ingredients/mL |
3h / 28h |
112 |
– |
NE |
|
78.125μg active ingredients/mL |
3h / 28h |
123 |
– |
NE |
|
Positive control |
3h / 28h |
62 |
– |
69.8*** |
|
Negative (solvent) control: 2 (v/v) % Distilled water
Positive control (-S9): Ethyl methanesulfonate, 0.4 µL/mL
Positive control (+S9): Cyclophosphamide, 6.0 µg/mL
NE: not evaluated
#: compared to the negative (solvent) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
Table 1: Results of the treated cultures
| Treatment condition | Test conc (µg/ml) | PE (%) |
RPE (%) |
SG | RSG (%) |
RTG (%) |
MF (x10-6) |
IMF (x10-6) |
MFsc (x10-6) | IMFsc (x10-6) |
| 3h without S9 | RMPI 10 | 90.69 | 100 | 9.57 | 100 | 100 | 123.38 | 0 | 32.53 | 0 |
| 8.192 | 88.31 | 97.38 | 9.22 | 96 | 93.81 | 151.2 | 27.82 | - | - | |
| 20.48 | 87.97 | 97 | 8.16 | 85 | 82.68 | 121.67 | -1.71 | - | - | |
| 51.2 | 85.37 | 94.13 | 9.9 | 103 | 97.34 | 148.5 | 25.12 | - | - | |
| 128 | 92.15 | 101.6 | 9.24 | 97 | 98.06 | 139.4 | 16.02 | - | - | |
| 320 | 66.77 | 73.62 | 8.39 | 88 | 64.51 | 179.89 | 56.51 | - | - | |
| 800 | 77.17 | 85.09 | 5.2 | 54 | 46.23 | 164.28 | 40.9 | - | - | |
| 2000 | 64.02 | 70.59 | 4.66 | 49 | 34.37 | 195.4 | 72.02 | 56.94 | 24.41 | |
| MMS (5.0) | 54.74 | 60.35 | 8.3 | 87 | 52.33 | 902.32 | 778.94 | 476.19 | 443.66 | |
| 3h with S9 | RMPI 10 | 90.69 | 100 | 8.33 | 100 | 100 | 132.43 | 0 | 27.98 | 0 |
| 8.192 | 93.94 | 103.59 | 7.82 | 94 | 97.24 | 133.16 | 0.73 | - | - | |
| 20.48 | 81.36 | 89.71 | 7.95 | 95 | 85.63 | 153.75 | 21.32 | - | - | |
| 51.2 | 90.69 | 100 | 8.27 | 99 | 99.35 | 139.79 | 7.35 | - | - | |
| 128 | 74.19 | 81.8 | 7.53 | 90 | 73.94 | 166.37 | 33.94 | - | - | |
| 320 | 69.66 | 76.8 | 8.18 | 98 | 75.42 | 196.6 | 64.17 | - | - | |
| 800 | 84.14 | 92.78 | 7.41 | 89 | 82.55 | 144.71 | 12.28 | - | - | |
| 2000 | 62.18 | 68.56 | 6.01 | 72 | 49.49 | 206.58 | 74.14 | 40.8 | 12.83 | |
| CP (3.0) | 59.61 | 65.73 | 6.8 | 82 | 53.71 | 883.04 | 750.61 | 534.65 | 506.68 | |
| 24h without S9 | RMPI 10 | 79.67 | 100 | 58.83 | 100 | 100 | 124.31 | 0 | 37.03 | 0 |
| 8.192 | 81.83 | 102.71 | 57.37 | 98 | 100.16 | 146.78 | 22.48 | - | - | |
| 20.48 | 68.69 | 86.22 | 56.22 | 96 | 82.4 | 153.47 | 29.16 | - | - | |
| 51.2 | 66.77 | 83.81 | 52.77 | 90 | 75.17 | 145.95 | 21.65 | - | - | |
| 128 | 76.69 | 96.26 | 55.66 | 95 | 91.09 | 137.47 | 13.16 | - | - | |
| 320 | 74.48 | 93.49 | 56.93 | 97 | 90.48 | 200.15 | 75.84 | - | - | |
| 800 | 60.45 | 75.87 | 49.02 | 83 | 63.22 | 218.1 | 93.8 | - | - | |
| 2000 | 57.94 | 72.73 | 37.57 | 64 | 46.44 | 133.39 | 9.08 | 34.38 | -2.64 | |
| MMS (5.0) | 50.21 | 63.03 | 39.1 | 66 | 41.9 | 949.37 | 825.07 | 610.5 | 573.47 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
The reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate was tested in vivo for chromosomal aberrations in a micronucleus test (mice).
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration (frequency of micronucleated polychromatic erythrocytes in bone marrow)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- between 2012-01-17 to 2012-04-17
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study
- Justification for type of information:
- As indicated in the EU REACH Regulation (Annex VIII), an in vivo mutagenicity test shall be considered in case of a positive result in any of the in-vitro genotoxicity studies in Annex VII or VIII. For the Reaction mass of TFSK/TFAK the in-vitro mutagenicity tests show negative results and do not trigger the performance of an in vivo study. It should however be noted that for the Reaction mass of TFSK/TFAK a new chemical substance notification in China has been prepared under the Regulation ‘Measures on the Environmental Management of New Chemical Substances' (Decree No. 7 of the Ministry of Environmental Protection of the P.R. China, also known as ‘China REACH’). Under this regulation the in vivo mutagenicity test is part of the standard data requirements for substances that are produced or imported in volumes > 10 t/y. For this reason an OECD 474 study was performed in 2012 at CiTox Lab. The results of the study are included in the dossier and serve as the valid test related to this endpoint.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Remarks:
- 2010-12-16
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Janvier Route des Chènes Secs B.P. 4105 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: approximately 7 weeks
- Weight at study initiation:
37.7 – 39.7 g (males, preliminary experiment I.)
28.2 – 30.2 g (females, preliminary experiment I.)
38.5 - 40.3 g (males, preliminary experiment II.)
26.3 - 27.9 g (females, preliminary experiment II.)
34.7 - 39.8 g (males, main test)
- Assigned to test groups randomly: [yes: ] The animals were assigned to their respective treatment groups by randomization based on body weights. Animals were randomly allocated to the negative and positive control groups based on the most recent actual body weight; SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups
- Fasting period before study:
- Housing: Group caging (5 animals/cage or 2 animals/cage) to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities. Cage type:II. type polypropylene/polycarbonate (37 x 22.5 x 18 cm). Bedding: Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-FASERN Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) was available to animals during the study.
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for mice and rats – breeding and maintenance" (Batch number: 683 5949, Expiry date: January 2012; and Batch number: 719 6627, Expiry date: May 2012) produced by ssniff Spezialdiäten GmbH (D-59494, Soest, Germany
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum
- Acclimation period: 6-14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 25.0°C
- Humidity (%): 31 – 69 %
- Air changes (per hr): 15 – 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
IN-LIFE DATES: From:2012-01-17 To:2012-01-23 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: Distilled water Batch No.:3450611 Source: TEVA Hungary Ltd. Expiry:June 2014 Storage conditions: Room temperature
- Justification for choice of solvent/vehicle: Based on the results of the preliminary solubility test, the test item was soluble in Distilled water
- Concentration of test material in vehicle: 2000, 1000 and 500 mg active ingredients/kg body weight/day
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in the distilled water for the treatment. The necessary amount of the test item was weighed into a calibrated volumetric flask; vehiclewas added and stirred to obtain homogenous formulations. For the purity conversion, the 36.5 % purity data of the two active ingredients (potassium trifluoromethanesulfinate and potassium trifluoroacetate) was taken into consideration. - Duration of treatment / exposure:
- One occasion
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Negative (vehicule) control: 15 males (In the negative control group unusually aggressive behaviour of one male animal was observed at the 24-hour necropsy. As this behaviour could have influenced the results of other four animals in the group (marked body weight loss and biting marks were recognized), additional five negative control animals were treated on using the random spare animals of the study as agreed by the Sponsor. These additional negative control animals were individually caged, but otherwise the experimental procedure was the same. Necropsy and slide preparation from these additional animals were performed 24-hours after treatment.
test item 500 mg/kg: 5 males
Test item 1000 mg/kg: 5 males
Test item 2000 mg/kg: 10 + 2 males (Two additional male mice were dosed in highest dose group (2000 mg/kg body weight/day) of the main experiment to replace any animals which die before the scheduled sacrifice time. Bone marrow smears were not prepared from additional mice as they were not used as replacement)
Positive control (cyclophosphamide): 5 males - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide Batch No.:079K1569 Supplier: Sigma-Aldrich Co.Expiry date: July 2012 Storage condition: Refrigerated (2-8 °C)
- Justification for choice of positive control(s): No data
- Route of administration: intraperitoneal injection
- Doses / concentrations: 60mg/kg using a dose volume of 10 mL/kg - Tissues and cell types examined:
- Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
In a preliminary study, 2000 and 1000 mg active ingredients/kg body weigh/day doses were tested in groups of 2 male and 2 female animals. All animals were free of clinical signs in the preliminary experiments except of one male showing piloerection.
Based on the results of the preliminary experiment, dose levels of 2000, 1000 and 500 mg active ingredients/kg body weight/day were selected for the micronucleus test. The main experiment was performed using male mice only as male mice were at least as sensitive to the test item toxic effects as female mice based on the results of the preliminary experiments.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Group Treatment mg active ingredients/kg bw/day* No. of Analysable Animals Period Between Treatment and Sampling time
1. Negative (vehicle) control -- 15 Males** 24 and 48 hours
2. Reaction mass of TFSK/TFAK, low dose 500 5 Males 24 hours
3. Reaction mass of TFSK/TFAK, mid dose 1000 5 Males 24 hours
4. Reaction mass of TFSK/TFAK, high dose 2000 10 + 2 Males*** 24 and 48 hours
5. Positive control (Cylophosphamide) 60 5 Males 24 hours
* Purity conversion was applied for the test item formulations (36.5 % purity data of the two active ingredients was taken into consideration).
** Additional five negative (vehicle) control animals were dosed in the main experiments (for further details see 6.2.).
***Two additional male mice were dosed in highest dose group (2000 mg/kg body weight/day) of the main experiment to replace any animals which die before the scheduled sacrifice time. Bone marrow smears were not prepared from additional mice as they were not used as replacement.
DETAILS OF SLIDE PREPARATION:
Bone marrow was obtained from two exposed femurs of mice immediately after sacrifice. The bone marrow was flushed out of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle into a sterile centrifuge tube. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (2 slides / animal). Slides were air-dried at room temperature for approximately 24 hours. Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Two slides per animal were stained with 10 % Giemsa solution for 20 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, coverslips were mounted on them.
METHOD OF ANALYSIS:
Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei were recorded in both types of erythrocytes.
OTHER: - Evaluation criteria:
- Criteria for a positive response:
The test item is considered to have shown genotoxic activity if statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, and the increases are dose-related. Historical control data could be taken into consideration when evaluating the biological significance of small increases.
Criteria for a negative response:
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historical control values.
Equivocal response:
Results which do not meet the criteria for a positive or negative response are considered to be equivocal. Further investigations or scoring of additional cells may be necessary in case of an equivocal result - Statistics:
- Kruskal Wallis test
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range:1000 and 2000 mg/kg active ingredient
- Solubility: soluble at 200 mg/mL
- Clinical signs of toxicity in test animals: All animals were free of clinical signs in the preliminary experiments except of one male showing piloerection.
- Evidence of cytotoxicity in tissue analyzed: No
- Rationale for exposure: Based on the results of the preliminary experiment, dose levels of 2000, 1000 and 500 mg active ingredients/kg body weight/day were selected for the micronucleus test.
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): See results table
- Appropriateness of dose levels and route: 500, 1000 and 2000 mg/kg active ingredient par oral route (gavage)
- Statistical evaluation: The group treated with 500 mg active ingredients/kg bw/day, which gave the highest individual number of micronuclei at 24h, were compared with the vehicle control group using the Kruskal Wallis test. This gave a value of H = 3.540. Comparison of the vehicle control data and the high dose of the test agent (2000 mg active ingredients/kg bw/day) at 48h gave a value of H = 0.565. Both H values are non-significant, giving a negative response.
The positive and negative control results were also compared, and gave a value of H = 7.211 (p<0.01). The positive control treatment therefore caused a large, statistically significant increase, demonstrating the sensitivity of the test system. - Conclusions:
- In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Reaction mass of TFSK/TFAK to mice at up to and including 2000 mg active ingredients/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.
- Executive summary:
In a NMRI mouse micronucleus assay on erythrocytes micronucleus test (cytoxlab study, 2012), from 5 to 15 males per dose were treated orally (gavage) with TFSK/TFAK at doses of 500, 1000 and 2000 mg/kg.
There were no mortality or signs of systemic toxicity during the study. No market effect of the test item treatment on the body weight of the mice was observed in the main test.
No biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were seen in mice treated with the test item, compared to the negative control values at any of the sampling time points.
In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Reaction mass of TFSK/TFAK to mice at up to and including 2000 mg active ingredients/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study
This study is classified as acceptable. This study was conducted comparably to the requirements of the Test Guideline OECD 474 for in vivo cytogenetic mutagenicity.
Reference
Micronucleus Data: Mice sacrificed 24 hours after dosing
Treatment |
ID |
Animal |
MNPCE/ |
PCE/1000 PCE+NCE |
Group 1 |
8465 |
12 |
1 |
307 |
Negative (vehicle) control |
8468 |
15 |
1 |
396 |
(Distilled water) |
8474 |
21 |
1 |
355 |
|
8483 |
30 |
0 |
339 |
|
8499 |
46 |
0 |
311 |
|
8457 |
4 |
1 |
308 |
|
8469 |
16 |
1 |
377 |
|
8480 |
27 |
2 |
317 |
|
8496 |
43 |
0 |
368 |
|
8501 |
48 |
0 |
414 |
Mean: |
|
|
0.7 |
349.2 |
SD: |
|
|
0.675 |
38.91 |
Group 2 |
8456 |
3 |
7 |
400 |
Reaction mass of |
8472 |
19 |
2 |
419 |
TFSK/TFAK |
8478 |
25 |
1 |
283 |
(500 mg/kg bw/day) |
8484 |
31 |
1 |
442 |
|
8491 |
38 |
1 |
437 |
Mean: |
|
|
2.4 |
396.2 |
SD: |
|
|
2.608 |
65.40 |
Group 3 |
8460 |
7 |
3 |
447 |
Reaction mass of |
8485 |
32 |
1 |
390 |
TFSK/TFAK |
8488 |
35 |
2 |
371 |
(1000 mg/kg bw/day) |
8500 |
47 |
5 |
490 |
|
8502 |
49 |
4 |
436 |
Mean: |
|
|
3.0 |
426.8 |
SD: |
|
|
1.581 |
47.31 |
Group 4 |
8462 |
9 |
0 |
367 |
Reaction mass of |
8466 |
13 |
4 |
453 |
TFSK/TFAK |
8467 |
14 |
3 |
405 |
(2000 mg/kg bw/day) |
8479 |
26 |
3 |
429 |
|
8494 |
41 |
3 |
341 |
Mean: |
|
|
2.6 |
399.0 |
SD: |
|
|
1.517 |
45.39 |
Group 5 |
8459 |
6 |
41 |
450 |
Positive Control |
8461 |
8 |
87 |
404 |
(Cyclophosphamide, |
8463 |
10 |
55 |
429 |
60 mg/kg bw/day) |
8470 |
17 |
35 |
426 |
|
8476 |
23 |
68 |
440 |
Mean: |
|
|
57.2 |
429.8 |
SD: |
|
|
21.005 |
17.27 |
MNPCE: Number of Micronucleated Polychromatic Erythrocytes referring to counts of 2000 PCE.
PCE: Polychromatic Erythrocyte
NCE: Normochromatic Erythrocyte
Micronucleus Data Mice sacrificed 48 hours after dosing
Treatment |
ID |
Animal |
MNPCE/ 2000 PCE |
PCE/1000 PCE+NCE |
Group 1 |
8455 |
2 |
1 |
406 |
Negative (vehicle) control |
8458 |
5 |
0 |
314 |
(Distilled water) |
8473 |
20 |
1 |
386 |
|
8486 |
33 |
0 |
273 |
|
8490 |
37 |
0 |
306 |
Mean: |
|
|
0.4 |
337.0 |
SD: |
|
|
0.548 |
56.45 |
Group 4 |
8471 |
18 |
0 |
321 |
Reaction mass of |
8481 |
28 |
2 |
432 |
TFSK/TFAK |
8482 |
29 |
0 |
416 |
(2000 mg/kg bw/day) |
8497 |
44 |
1 |
430 |
|
8498 |
45 |
1 |
353 |
Mean: |
|
|
0.8 |
390.4 |
SD: |
|
|
0.837 |
50.42 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro gene mutation study in bacteria
The Reaction mass of TFSK/TFAK was examined for mutagenic activity in the Ames test (OECD guideline 471 and GLP compliant) using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA, in the absence and presence of metabolic activation. All strains were treated with seven concentrations of the test substance, ranging from 5 to 5000 µg/plate. Negative controls and positive controls were run simultaneously with the test substance. The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean number of revertant colonies. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed. In both the absence and presence of S9-mix in all strains, Reaction mass of TFSK/TFAK did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.
In vitro gene mutation study in mammalian cells
In a study according to OECD guideline 490 and in compliance with GLP, the Reaction mass of TFSK/TFAK was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells. No test-substance induced increase in the number of mutations was observed when tested up to limit concentration in the presence and absence of metabolic activation. The result of the first experiment, 3-hour exposure, with and without metabolic activation, was confirmed in a second experiment, 24-hour exposure without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.
In vitro cytogenicity / chromosome aberration study in mammalian cells
The Reaction mass of TFSK/TFAK was tested In a Chromosome Aberration Assay using Chinese hamster V79 lung cells performed according to OECD Guideline 473 and GLP. Two independent assays were conducted in the presence and absence of metabolic activation. The negative and positive controls gave valid results. No induction of chromosome aberrations in Chinese hamster V79 cells was observed following treatment with Reaction mass of TFSK/TFAK up to 5000 μg active ingredients/mL concentration; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.
In vivo Micronucleus test
A GLP-compliant erythrocyte micronucleus test was performed according OECD guideline 474. Four groups of 5 to 15 male animals were dosed via oral gavage with vehicle or with the Reaction mass of TFSK/TFAK at doses of 500, 1000 and 2000 mg/kg bw. A positive control group (cyclophosphamide) was included. It was concluded that, under the conditions used in this study, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Reaction mass of TFSK/TFAK to mice at up to and including 2000 mg active ingredients/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.
Justification for classification or non-classification
The reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate was tested in vitro in an Ames test and in a chromosomal aberration test and in vivo in a micronucleus test. All these tests were negative. Based on these results, the reaction mass of TFAK/TFSK is not classified for genetic toxicity.
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