Registration Dossier
Registration Dossier
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EC number: 903-945-5 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Since all the available in vitro tests showed negative results, the reaction mass of chlorodifluoroacetic acid and trifluoroacetic acid is not to be considered as mutagenic.
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 07 july 2003 to 10 oct 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP study, OECD 471 compliant.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix rat liver fraction treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 0, 312.5, 625, 1250, 2500 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: NaCl (0.9%)
- Justification for choice of solvent/vehicle: the most appropriate for the solubility of CDFA, and among recommended vehicles by the guideline. - Untreated negative controls:
- no
- Remarks:
- vehicle served as negative control
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide, 9-Aminoacridine, 2-Nitroflurene, Mytomycin C, 4-Nitroquinoline-1oxide, 2-Anthramine
- Remarks:
- Details in test conditions
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) for preliminary toxicity test and for the first experiment and in the second experiment without S9 mix; preincubation in the second experiment with S9 mix.
DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 to 72 hours at 37°C
NUMBER OF REPLICATES: 3 - Evaluation criteria:
- A reproductible 2-fold increase (for the TA 98, TA 100, TA 102, and E. coli WP2 uvrA strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
- 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 examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- thinning of the bacterial lawn only in TA 100 strain
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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 examined
- Positive controls validity:
- valid
- 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 examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING STUDY:
With a treatment volume of 100 µL/plate, the dose levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.
No noteworthy toxicity was noted towards the four satrins used, with and without S9 mix.
COMPARISON WITH HISTORICAL CONTROL DATA: the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. - Conclusions:
- Chlorodifluoroacetic acid did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli in the absence and presence of metabolic activation.
- Executive summary:
In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 102, TA 1535, TA 1537 of S. typhimurium and E. coli E. coli WP2 uvr A were exposed to Chlorodifluoroacetic acid at concentrations of 0 to 5000 µg/plate in the presence and absence of mammalian metabolic activation (rat liver).
The plate incorporation method was used for preliminary toxicity test and for the first experiment and in the second experiment without S9 mix. Pre-incubation was used in the second experiment with S9 mix.
The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of increase of mutant colonies. Chlorodifluoroacetic acid did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 11 aug 2002 to 26 march 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP study, OECD 471 compliant.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix rat liver fraction treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 0, 0.1, 1, 10, 100, 1000 and 10000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: NaCl
- Justification for choice of solvent/vehicle: solvent usually used in this type of study, and the most appropriate for the solubility of CDFA - Untreated negative controls:
- no
- Remarks:
- vehicle served as negative control
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide, 9-Aminoacridine, 2-Nitroflurene, 2-Aminoanthracene, 1-Ethyl-3-Nitro- -1-Nitroguanidine
- Remarks:
- Details in test conditions
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) for preliminary toxicity test and for the first experiment and in the second experiment without S9 mix; preincubation in the second experiment with S9 mix.
DURATION
- Exposure duration: 48 hours at 37°C
NUMBER OF REPLICATES: 3 - Evaluation criteria:
- The test item is considered to have caused a positive response in the assay if at least at one test item dose exhibits a reproductile and statistically significant increase (p=<0.5) in the number of mutants over its concurrent negative control.
The test item is considered to have caused a positive dose response in the assay if a dose-related increase in the number of mutants with r>=0.95 (obtained from the Linear Regression data analysis) is observed, with at least one test article dose showing a reproducible and statistically significant increase (=<0.05) over the negative control. - Statistics:
- Statistical program by R.J. Tallarida and R.B. Murray named Pharmacologic Calculations Procedure, ANOVA (analysis of variance) and Newman-Keuls Test foe confirmation of pairwise comparisons. This statistical method determines if there is a significant (p=<0.05) increase in the mutation frequency of the test article compared to the negative control article.
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING STUDY:
With a treatment volume of 100 µL/plate, the dose levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.
No noteworthy toxicity was noted towards the four satrins used, with and without S9 mix.
COMPARISON WITH HISTORICAL CONTROL DATA: the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. - Conclusions:
- Chlorodifluoroacetic acid did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli in presence and absence of metabolic activation.
- Executive summary:
The Salmonella typhimurium and Escherichia coli reverse mutation assay (Ames test) evaluated the potential of the test article, Chlorodifluoroacetic acid (CDFA) to induce histidine reversion and tryptophan reversion in the genomes of these respective organisms.
This direct plate incorporation assay was conducted in triplicate with four strains of S. typhimurium which were TA98, TA100, TA1535 and TA1537 and one strain of E. coli which was WP2 in the presence and absence of an exogenous mammalian activation system, at doses from 0 to10000 µg/plate.
All positive controls exhibit a statistically significant increase in the number of revertant colonies as compared to the corresponding negative control, validating the functioning of the assay.
No statistically increase was observed with CDFA at all tested concentrations. A confirmatory assay with a direct plate incorporation method was performed in order to verify the results.
Based on these test conditions, CDFA was not considered as mutagenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1986-03-04 to 1986-03-25
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- The study was performed similarly to the OECD (No. 471) with acceptable restrictions and was in compliance to the GLP. Sodium Trifluoroacetate was tested instead of trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of TFA (pH=0.45).
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- : only the 2-Anthramine is used as a positive control for the efficacy of S9 mix. Strain S. typhimurium TA102 or E.coli WP2 were not used. No data on the composition of the substance tested.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Trifluoroacetic acid (TFA) is a strong acid. Testing with the neutral salt sodium trifluoroacetate was considered appropriate to avoid cytotoxic effects on bacteria due to the extreme acid pH of TFA (pH=0.45).
- Target gene:
- Each strains derived from S. thyphimurium LT2 contains one mutation in the histidine operon, resulting in a requirement for histidine. See details in Table 7.6.1/1.
- Species / strain / cell type:
- S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- other: additional mutation in uvrB and rfa genes. The plasmid pKM101 was present in TA98 and TA100 in order to increase the sensibility of these strains to some mutagens. See details in Table 7.6.1/1.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction was obtained from the liver of Sprague-Dawley male rats treated by the intraperitoneal route with Aroclor 1254 (500 mg/kg) dissolved in maize oil.
- Test concentrations with justification for top dose:
- Preliminary test: 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 mg/plate.
Mutagenicity experiments: 0.1, 0.5, 1, 5 and 10 mg/plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
The susbtance is dissolved in DMSO at the highest concentration of 100 mg/mL. The succesive dilutions were performed from this initial solution. - Untreated negative controls:
- yes
- Remarks:
- sterility controls for the substance's solution, the solvent and the S9 mix.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 9-Aminoacridine (TA1537), 2-Nitrofluorene (TA98 and TA1538), Methyl methanesulphonate (TA 100) and Ethyl methanesulphonate (TA1535) for without S9 mix efficacy control. 2-Anthramine for with S9 mix efficacy control.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
direct incorporation method: 100 µL of the DMSO diluted substance were incorporated in glass tubes. Then were added the bacteria (0.1 mL), the S9 fraction (0.5 mL) in the case of test with metabolic activation and the agar. After agitation the mix was plated on a Petri plate containing Vogel and Bonner medium.
DURATION
- Preincubation period: no data
- Exposure duration: 48 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 3 plates/dose/strain. Two independent experiments were performed.
NUMBER OF CELLS EVALUATED: no data
DETERMINATION OF CYTOTOXICITY
- Method: no data
OTHER EXAMINATIONS:
- Determination of polyploidy: not required
- Determination of endoreplication: not required
- Other:
OTHER: - Evaluation criteria:
- A reproducible 2-fold increase in the number of revertants compared with the vehicle controls was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
- Statistics:
- no data
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS: not applicable
RANGE-FINDING/SCREENING STUDIES: a preliminary study was performed on the TA100 strain without metabolic activation. The tested concentrations of the substance were 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 mg/plate. No cytotoxicity in terms of decrease of the number of revertants or effect on the bacterial lawn was observed.
COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants for the vehicle and positive controls was similar to the historical data.
ADDITIONAL INFORMATION ON CYTOTOXICITY: no additional information - Conclusions:
- Under the test conditions, the test item Sodium Trifluoroacetate did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
- Executive summary:
In a reverse gene mutation assay in bacteria, performed similarly to the OECD No.471 guideline, strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium were exposed to Sodium Trifluoroacetate diluted in DMSO at concentrations of 0.1, 0.5, 1.0, 5.0 and 10 mg/plate in the presence and absence of mammalian metabolic activation (fraction of S9 from the liver of Sprague-Dawley male rats treated by intraperitoneal injection with Aroclor 1254 dissolved in maize oil). The method of direct incorporation was used in this study.
Sodium Trifluoroacetate was tested up to limit concentration recommended in the guideline (5 mg/plate).
The positive controls induced the appropriate responses in the corresponding strains.
Under the test conditions, the test item Sodium trifluoroacetate did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium as there was no evidence of induced mutant colonies over background.
In this study Sodium Trifluoroacetate was used instead of Trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of the TFA.
Therefore, Trifluoroacetic acid is not considered as mutagenic in the bacterial reverse mutation test.
This study is considered as acceptable as it satisfied the main criteria of the OECD guideline No. 471.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 14 oct 2011 to 12 nov 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP study, OECD 473 compliant
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 15 august 2011
- 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):
- V79: Chinese hamster lung, male
ECACC: 86041102
Lot. Number: 05F013
Supplier: ECACC (European Collection of Cells Cultures)
The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks are kept in a freezer at -80 ± 10C. The stock was checked for mycoplasma infection.
Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution will be used for cell detachment to subculture. The laboratory cultures will be maintained in 75 cm2 plastic flasks at 37 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. The V79 cells for this study will be grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1% Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 g/mL amphoptericin-B) and 10 (v/v) % heat-inactivated foetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium will be reduced to 5% (DMEM-5). - Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9 mix
- Test concentrations with justification for top dose:
- Experiment 1: 5000; 4900; 4800; 4700; 4600; 4500; 4400; 4300; 4200; 4100; 4000; 2000 and 1000 μg/mL.
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.
Experiment 2: 5000; 4900; 4800; 4700; 4600; 4500; 4400; 4300; 4200; 4100; 4000; 2000; 1000 and 500 μg/mL.
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.
Experiment 3: 5000; 4500; 4000; 3000; 2500; 2000; 1500; 1000 and 500 μg/mL.
A 3-hour treatment without metabolic activation (in the absence of S9-mix) was performed. Sampling was performed 20 hours after the beginning of the treatment. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: most appropriate for the solubility of CDFA, and recommended by the guideline. - Untreated negative controls:
- yes
- Remarks:
- Solvent served as negative controls
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Ethyl methanesulfonate (1 µL/mL) and Cyclophosphamide (6 µg/mL
- Details on test system and experimental conditions:
- Preparation of Chromosomes:
Two to 2.5 hours prior to harvesting, cell cultures were treated with colchicine (0.2 μg/mL). The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried. The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted.
Toxicity and Concentration Selection:
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test. In this Preliminary Toxicity Test, two separate assays were performed. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time. In Assay B, cells were treated for 3 hours in the presence of S9-mix and for 20 hours in the absence of S9-mix with a 28-hour harvesting time. The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.
At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (solvent) control as % relative survival.
For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (solvent) control as % relative survival. - Evaluation criteria:
- The assay was considered valid, if the following criteria are met:
- The solvent control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.
The test item was considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are 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.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 2000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA:
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.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
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; 4900; 4800; 4700; 4600; 4500; 4400; 4300; 4200; 4100; 4000; 2000 and 1000 μg/mL.
In Assay 1, no insolubility was observed in the final treatment medium at the end of the treatment either in the presence or absence of the metabolic activation system. No large changes were detected in osmolality, but large pH changes were detected at the highest examined concentrations due to the test item’s chemical nature. Based on the observed cytotoxicity results, concentrations of 4500; 4000; 2000 and 1000 μg/mL (a total of four) were chosen for evaluation in the experiment without metabolic activation; and concentrations of 4000; 2000 and 1000 μg/mL (a total of three) were chosen for evaluation in case of the experiment with metabolic activation. None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations in case of the experiment with metabolic activation. In the experiment without metabolic activation, one concentration (2000 μg/mL) showed a marginal increase in the number of structural chromosome aberrations.
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; 4900; 4800; 4700; 4600; 4500; 4400; 4300; 4200; 4100; 4000; 2000; 1000 and 500 μg/mL.
In Assay 2, similarly to the first experiment, no insolubility was 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 osmolality, but large pH changes were detected at the highest examined concentrations similarly to the previous assay. Based on the observed cytotoxicity results, concentrations of 4000; 2000 and 1000 μg/mL (a total of three) were chosen for evaluation in case of the experiment without metabolic activation; and concentrations of 4500; 4000; 2000 and 1000 μg/mL (a total of four) were chosen for evaluation in case of the experiment with metabolic activation.
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 3, a 3-hour treatment without metabolic activation (in the absence of S9-mix) was performed. Sampling was performed 20 hours after the beginning of the treatment. The examined concentrations of the test item were 5000; 4500; 4000; 3000; 2500; 2000; 1500; 1000 and 500 μg/mL.
In Assay 3, no insolubility was observed in the final treatment medium at the end of the treatment. No large changes were detected in osmolality, but large pH changes were detected at the highest examined concentrations similarly to the previous assays. Based on the observed cytotoxicity results and the results of Assay 1, concentrations of 4000; 3000; 2000 and 1000 μg/mL (a total of four) were chosen for metaphase analysis. No increase in the number of structural chromosome aberrations in the evaluated test item treated samples was observed in this experiment. - Conclusions:
- In conclusion, no repeatable induction of chromosome aberrations in Chinese hamster V79 cells was observed following treatment with Chlorodifluoroacetic acid up to the cytotoxic concentrations; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study. Therefore, Chlorodifluoroacetic acid is considered non-clastogenic in this test system.
- Executive summary:
The test item Chlorodifluoroacetic acid was tested for potential clastogenic activity using the Chromosome Aberration Assay. The study (2012) included two Concentration Selection Cytotoxicity Assays and three Chromosome Aberration Assays at doses from 500 to 5000 µg/plates.
The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.
Treatment with the test item did not resulted in a statistically and biologically significant increase in the frequency of the cells with structural chromosome aberrations either in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats.
In conclusion, no repeatable induction of chromosome aberrations in Chinese hamster V79 cells was observed following treatment with Chlorodifluoroacetic acid up to the cytotoxic concentrations; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study. Therefore, Chlorodifluoroacetic acid is considered non-clastogenic in this test system.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- GLP study in accordance with international guidelines. Sodium Trifluoroacetate was tested instead of trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of TFA (pH = 0.45).
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- minor deviations, did not affect the integrity of the study
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Trifluoroacetic acid (TFA) is a strong acid. Testing with the neutral salt sodium trifluoroacetate was considered appropriate to avoid cytotoxic effects on bacteria due to the extreme acid pH of TFA (pH=0.45).
- Species / strain / cell type:
- lymphocytes: human (female)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 µg/mL gentamycin. Phytohaemagglutinin is included in the medium at a concentration of approximately 2% of culture.
Blood from three healthy, non-smoking female volunteers was used for each experiment of this study.
No volunteer was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 1.5 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within two days of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction from male Sprague-Dawley rats induced Arochlor 1254
- Test concentrations with justification for top dose:
- First test
Without S9 mix - 3 hours treatment, 17 hours recovery: 1000; 1200 and 1360 µg/mL
With S9 mix - 3 hours treatment, 17 hours recovery: 1000; 1200 and 1360 µg/mL
Second test
Without S9 mix - 20 hours continuous treatment: 750; 1200 and 1360 µg/mL
With S9 mix - 3 hours treatment, 17 hours recovery: 750; 1200 and 1360 µg/mL - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without S9: 4-Nitroquinoline-1-oxide (2.5 µg/mL); with S9: cyclophosphamide (20 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 48 h
- Exposure duration: First experiment 3 h
Second experiment 20 h (withour S9) / 3 h (with S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h
SPINDLE INHIBITOR (cytogenetic assays): colchicine (final concentration: 1 µg/mL)
STAIN (for cytogenetic assays): 4% (v/v) Giemsa
NUMBER OF REPLICATIONS: 2 (4 for vehicle)
NUMBER OF CELLS EVALUATED: at least 1000 cells counted where possible
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Determination of hyperdiploidy : yes - Evaluation criteria:
- The test substance will be considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeds the historical negative control (normal) range is observed in both replicate cultures.
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) is observed (p < 0.05).
3. There is a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article will be considered as positive in this assay if all of the above criteria are met.
The test article will be considered as negative in this assay if none of the above criteria are met.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Evidence of a concentration-related effect is considered useful but not essential in the evaluation of a positive result. Biological relevance will be taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations. Analysis of additional cells from vehicle or treated cultures or further experimental work may be deemed necessary to aid evaluation of the data. - Statistics:
- The statistical method used will be Fisher's exact test. Probability values of p < 0.05 will be accepted as significant.
The proportions of aberrant cells in each replicate will be used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. Probability values of p < 0.05 will be accepted as significant. - Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA: yes
- Conclusions:
- Under the test conditions, Sodium trifluoroacetate (56%) did not induce chromosome aberrations in cultured human peripheral blood lymphocytes in the presence and the absence of S-9 when tested up to a maximum concentration equivalent to 10 mM.
- Executive summary:
In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to Sodium trifluoroacetate 55.6 % aqueous solution. In the fist experiment cells were exposed both in the presence and absence of S-9 to concentrations of 0, 1000, 1250, 1360 µg/mL for 3 hours and sampled at 20 hours after the beginning of treatment. In the second experiment, cells were exposed continuously for 20 hours in the absence of S-9 (0, 750, 1200 and 1360 µg/mL) or 3 hours in the presence of S-9 (0, 1050, 1200 and 1360 µg/mL).
Positive controls induced the appropriate response. Chromosome aberrations were not induced over background at any tested concentrations in the absence and the presence of activation system in both experiments.
Under the test conditions, Sodium trifluoroacetate (56%) was not clastogenic in human lymphocytes exposed in vitro.
In this study Sodium Trifluoroacetate was used instead of Trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of the TFA
Therefore, Trifluoroacetic acid is not considered as clastogenic in humans cells exposed in vitro.
This study is considered as acceptable and satisfies the requirement for the cytogenicity endpoint.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- GLP study in accordance with international guidelines. Sodium Trifluoroacetate was tested instead of trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of TFA (pH=0.45).
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Specific details on test material used for the study:
- Trifluoroacetic acid (TFA) is a strong acid. Testing with the neutral salt sodium trifluoroacetate was considered appropriate to avoid cytotoxic effects on bacteria due to the extreme acid pH of TFA (pH=0.45).
- Target gene:
- The hypoxanthine-guanine phosphoribosyl transferase (hprt) gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 10 medium prepared as follow:
Horse serum (heat inactivated): 10% v/v
Penicillin/Streptomycin: 100 units/mL / 100 µg/L
Amphotericin B: 2.5 µg/mL
Pluronic: 0.5 mg/L
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9: Aroclor 1254 induced rat liver post mitochondrial fraction
- Test concentrations with justification for top dose:
- Experiment 1 (+/- S9): 200, 400, 600, 800, 100, 1200 and 1360 µg/mL.
Experiment 2 (+/- S9): 150, 300, 500, 700, 900, 1100 and 1360 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitroquinoline-1-oxide (NQO) 0.1 and 0.15 µg/mL; Benzo[a]pyrene (BP) 2 and 3 µg/mL
- Remarks:
- no remarks
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 hours incubation at 37±1°C
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of at least 7 days during which the hprt- mutation will be expressed.
- Selection time (if incubation with a selection agent): one to two weekss
- Fixation time (start of exposure up to fixation or harvest of cells): no data
SELECTION AGENT (mutation assays): thio-6-guanine (6TG)
NUMBER OF REPLICATIONS: duplicate
NUMBER OF CELLS EVALUATED: no data
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth - Evaluation criteria:
- For valid data, the test article was considered to be mutagenic in this assay if:
1. The mutant frequency at one or more concentrations is significantly greater than that of the negative control (p<0.05)
2. There is a significant concentration relationship as indicated by the linear trend analysis (p<0.05)
3. The effects described above are reproducible.
Results which only partially satisfy the above criteria will be dealt with on a case by case basis. Positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Extreme caution will be exercised with positive results obtained at levels of RS lower than 10%. - Statistics:
- Statistical significance of mutant frequencies will be carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) will be compared with the LMF from each test article treatment, and secondly the data will be checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: In the range-finder, there was no evidence of marked toxicity at concentrations up to and including 1360 µg/mL (equivalent to 10 mM) in the absence and presence of S-9. See table 7.6.1/3.
COMPARISON WITH HISTORICAL CONTROL DATA: yes
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment 1 the highest concentration selected, 1360 µg/mL, gave 97% and 84% RS in the absence and presence of S 9, respectively.
In experiment 2 the highest concentration selected, 1360 µg/mL, gave 80% and 112% RS in the absence and presence of S 9, respectively. - Conclusions:
- Sodium trifluoroacetate (55.6% aq solution) was not mutagenic in the MLA when tested up to 10 mM in the absence and presence of S-9.
- Executive summary:
In an in vitro mammalian cell gene mutation test performed according to the OECD test guideline No. 476 and in compliance with GLP, Sodium trifluoracetate as a 55.6% aqueous solution was assayed for its ability to induce mutation at the hprt locus (thio-6 -guanine resistance) in mouse lymphoma L5178Y cells.
The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post‑mitochondrial fraction (S‑9).
In the cytotoxicity range-finder experiment, 6 concentrations were tested in the absence and presence of S-9, ranging from 42.5 to 1360 µg/mL. There was no evidence of marked toxicity at concentrations up to and including 1360 µg/mL (equivalent to 10 mM) in the absence and presence of S-9.
In Experiment 1 seven concentrations, ranging from 200 to 1360 µg/mL, were tested in the absence and presence of S‑9.
In Experiment 2 seven concentrations, ranging from 150 to 1360 µg/mL were tested in the absence and presence of S-9.
When tested up to 1360 µg/mL (10 mM) in the absence and presence of S-9 in Experiments 1 and 2, no significant increases in mutant frequency were observed at any concentration analysed and there were no significant linear trends.
In the presence of S-9 in Experiment 1, the positive controls did not meet the acceptance criteria stated in the protocol. However, both positive control concentrations showed clear increases in mutant frequency which were outside the historical negative control ranges generated by the last 20 studies, updated at the time of each experiment. The data were therefore considered acceptable and valid on this basis.
Negative controls were valid for both experiments.
It is therefore conclude that sodium trifluoroacetate (55.6% aq solution) was not mutagenic when tested up to 10 mM in the absence and presence of S-9.
In this study Sodium Trifluoroacetate was used instead of Trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of the TFA (see §4.2).
Therefore, Trifluoroacetic acid is not considered as mutagenic in the mammalian cell gene mutation test.
Referenceopen allclose all
First experiment: Direct plate incorporation method
Strain |
Compound |
Dose level (µg/plate) |
Metabolic activation |
Mean revertant colony counts |
TA 1535 |
0.9% NaCl |
0 |
- S9 mix |
14 |
CDFA |
312.5 |
15 |
||
625 |
15 |
|||
1250 |
10 |
|||
2500 |
15 |
|||
5000 |
11 |
|||
NAN3 |
1 |
530 |
||
0.9% NaCl |
0 |
+ S9 mix |
14 |
|
CDFA |
312.5 |
13 |
||
625 |
13 |
|||
1250 |
14 |
|||
2500 |
14 |
|||
5000 |
13 |
|||
2AM |
2 |
251 |
||
TA 100 |
0.9% NaCl |
0 |
- S9 mix |
97 |
CDFA |
312.5 |
98 |
||
625 |
117 |
|||
1250 |
101 |
|||
2500 |
113 |
|||
5000 |
102 |
|||
NAN3 |
1 |
615 |
||
0.9% NaCl |
0 |
+ S9 mix |
119 |
|
CDFA |
312.5 |
129 |
||
625 |
119 |
|||
1250 |
110 |
|||
2500 |
114 |
|||
5000 |
101 |
|||
2AM |
2 |
1750 |
||
TA 102 |
0.9% NaCl |
0 |
- S9 mix |
393 |
CDFA |
312.5 |
367 |
||
625 |
426 |
|||
1250 |
375 |
|||
2500 |
400 |
|||
5000 |
368 |
|||
NAN3 |
1 |
1332 |
||
0.9% NaCl |
0 |
+ S9 mix |
582 |
|
CDFA |
312.5 |
561 |
||
625 |
571 |
|||
1250 |
519 |
|||
2500 |
588 |
|||
5000 |
472 |
|||
2AM |
2 |
1768 |
Strain |
Compound |
Dose level (µg/plate) |
Metabolic activation |
Mean revertant colony counts |
WP2 uvrA |
0.9% NaCl |
0 |
- S9 mix |
30 |
CDFA |
312.5 |
31 |
||
625 |
32 |
|||
1250 |
38 |
|||
2500 |
31 |
|||
5000 |
29 |
|||
NAN3 |
1 |
219 |
||
0.9% NaCl |
0 |
+ S9 mix |
37 |
|
CDFA |
312.5 |
38 |
||
625 |
33 |
|||
1250 |
25 |
|||
2500 |
32 |
|||
5000 |
32 |
|||
2AM |
2 |
480 |
||
TA 1537 |
0.9% NaCl |
0 |
- S9 mix |
5 |
CDFA |
312.5 |
6 |
||
625 |
7 |
|||
1250 |
9 |
|||
2500 |
5 |
|||
5000 |
4 |
|||
NAN3 |
1 |
280 |
||
0.9% NaCl |
0 |
+ S9 mix |
7 |
|
CDFA |
312.5 |
8 |
||
625 |
6 |
|||
1250 |
7 |
|||
2500 |
10 |
|||
5000 |
7 |
|||
2AM |
2 |
108 |
||
TA 98 |
0.9% NaCl |
0 |
- S9 mix |
21 |
CDFA |
312.5 |
20 |
||
625 |
21 |
|||
1250 |
21 |
|||
2500 |
22 |
|||
5000 |
30 |
|||
NAN3 |
1 |
211 |
||
0.9% NaCl |
0 |
+ S9 mix |
28 |
|
CDFA |
312.5 |
27 |
||
625 |
27 |
|||
1250 |
30 |
|||
2500 |
27 |
|||
5000 |
30 |
|||
2AM |
2 |
1699 |
Second experiment : Direct plate incorporation method (without S9 mix) and preincubation method (with S9 mix)
Strain |
Compound |
Dose level (µg/plate) |
Metabolic activation |
Mean revertant colony counts |
TA 1535 |
0.9% NaCl |
0 |
- S9 mix |
12 |
CDFA |
312.5 |
17 |
||
625 |
12 |
|||
1250 |
13 |
|||
2500 |
13 |
|||
5000 |
14 |
|||
NAN3 |
1 |
607 |
||
0.9% NaCl |
0 |
+ S9 mix |
12 |
|
CDFA |
312.5 |
15 |
||
625 |
18 |
|||
1250 |
13 |
|||
2500 |
13 |
|||
5000 |
11 |
|||
2AM |
2 |
147 |
||
TA 100 |
0.9% NaCl |
0 |
- S9 mix |
93 |
CDFA |
312.5 |
108 |
||
625 |
107 |
|||
1250 |
128 |
|||
2500 |
123 |
|||
5000 |
123 |
|||
NAN3 |
1 |
640 |
||
0.9% NaCl |
0 |
+ S9 mix |
119 |
|
CDFA |
312.5 |
132 |
||
625 |
131 |
|||
1250 |
135 |
|||
2500 |
128 |
|||
5000 |
138 |
|||
2AM |
2 |
1539 |
||
TA 102 |
0.9% NaCl |
0 |
- S9 mix |
399 |
CDFA |
312.5 |
394 |
||
625 |
420 |
|||
1250 |
427 |
|||
2500 |
401 |
|||
5000 |
437 |
|||
NAN3 |
1 |
1342 |
||
0.9% NaCl |
0 |
+ S9 mix |
483 |
|
CDFA |
312.5 |
460 |
||
625 |
452 |
|||
1250 |
453 |
|||
2500 |
490 |
|||
5000 |
438 |
|||
2AM |
2 |
2122 |
Strain |
Compound |
Dose level (µg/plate) |
Metabolic activation |
Mean revertant colony counts |
WP2 uvrA |
0.9% NaCl |
0 |
- S9 mix |
29 |
CDFA |
312.5 |
32 |
||
625 |
24 |
|||
1250 |
20 |
|||
2500 |
19 |
|||
5000 |
32 |
|||
NAN3 |
1 |
468 |
||
0.9% NaCl |
0 |
+ S9 mix |
42 |
|
CDFA |
312.5 |
39 |
||
625 |
39 |
|||
1250 |
47 |
|||
2500 |
30 |
|||
5000 |
38 |
|||
2AM |
2 |
221 |
||
TA 1537 |
0.9% NaCl |
0 |
- S9 mix |
6 |
CDFA |
312.5 |
14 |
||
625 |
6 |
|||
1250 |
6 |
|||
2500 |
14 |
|||
5000 |
6 |
|||
NAN3 |
1 |
285 |
||
0.9% NaCl |
0 |
+ S9 mix |
7 |
|
CDFA |
312.5 |
6 |
||
625 |
8 |
|||
1250 |
6 |
|||
2500 |
10 |
|||
5000 |
9 |
|||
2AM |
2 |
142 |
||
TA 98 |
0.9% NaCl |
0 |
- S9 mix |
26 |
CDFA |
312.5 |
29 |
||
625 |
30 |
|||
1250 |
32 |
|||
2500 |
24 |
|||
5000 |
26 |
|||
NAN3 |
1 |
172 |
||
0.9% NaCl |
0 |
+ S9 mix |
23 |
|
CDFA |
312.5 |
26 |
||
625 |
29 |
|||
1250 |
25 |
|||
2500 |
22 |
|||
5000 |
16 |
|||
2AM |
2 |
1675 |
Table 7.6.1/4:Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in first test (direct plate incorporation method)
TFAS Concentration |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
TA 1538 |
|||||
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
|
0* |
12.7 |
5.5 |
10.7 |
2.9 |
28.0 |
3.6 |
86.0 |
7.8 |
18.3 |
5.7 |
0.1 |
12.7 |
4.9 |
16.0 |
2.6 |
20.3 |
4.0 |
84.0 |
7.5 |
20.0 |
7.0 |
0.5 |
14.3 |
7.6 |
10.7 |
4.1 |
17.0 |
1.7 |
83.0 |
3.6 |
15.3 |
1.1 |
1 |
15.3 |
3.5 |
10.3 |
1.1 |
17.7 |
5.5 |
86.0 |
6.9 |
17.0 |
2.6 |
5 |
14.0 |
2.6 |
11.7 |
4.0 |
16.3 |
1.5 |
100.3 |
10.7 |
23.0 |
10.5 |
10 |
18.7 |
4.0 |
15.3 |
4.1 |
25.7 |
2.3 |
102.3 |
4.9 |
17.0 |
1.7 |
Positive control** |
5723.0 |
- |
989.0 |
- |
264.5 |
- |
301.0 |
- |
199.0 |
- |
*Solvent control = negative control: 100 µL DMSO
**Mutagens positive controls:
- 2.N.F. (0.001 mg/plate) in TA98 and TA1538 strains
- M.M.S. (0.1 mg/plate) in TA100 strain
- E.M.S. (10 mg/plate) in TA1535 strain
- 9 A.A. ( 0.05 mg/plate) in TA 1537 strain
Table 7.6.1/5:Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (S9 mix) in first test (direct plate incorporation method)
TFAS Concentration |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
TA 1538 |
|||||
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
|
0* |
15.7 |
8.1 |
12.0 |
2.6 |
27.3 |
8.5 |
107.7 |
13.0 |
32.0 |
2.6 |
0.1 |
20.3 |
2.3 |
11.3 |
1.1 |
29.0 |
5.0 |
106.3 |
16.0 |
36.0 |
7.0 |
0.5 |
19.3 |
1.5 |
19.3 |
6.6 |
28.0 |
1.0 |
99.3 |
3.0 |
27.7 |
4.1 |
1 |
15.3 |
4.0 |
12.3 |
2.9 |
31.3 |
2.5 |
94.7 |
6.6 |
30.0 |
9.1 |
5 |
15.0 |
3.0 |
15.0 |
3.0 |
31.7 |
5.8 |
98.7 |
12.3 |
28.0 |
3.0 |
10 |
17.3 |
3.2 |
11.3 |
0.6 |
26.7 |
6.5 |
109.3 |
13.0 |
26.7 |
11.0 |
Positive control** |
225.0 |
- |
150.5 |
- |
1003.0 |
- |
1453.0 |
- |
831.0 |
- |
*Solvent control = negative control: 100 µL DMSO
**Mutagens positive controls:
- 2.A. (0.002 mg/plate)
Table 7.6.1/6:Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in second test (plate incorporation method)
TFAS Concentration |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
TA 1538 |
|||||
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
|
0* |
9.7 |
4.5 |
12.0 |
2.0 |
33.7 |
3.0 |
99.7 |
19.5 |
20.0 |
4.0 |
0.1 |
4.3 |
2.1 |
8.3 |
3.0 |
29.7 |
4.1 |
103.0 |
8.6 |
20.3 |
5.8 |
0.5 |
6.0 |
1.0 |
6.0 |
1.0 |
27.7 |
2.3 |
109.3 |
3.2 |
18.7 |
1.1 |
1 |
9.0 |
3.0 |
10.3 |
1.1 |
25.0 |
6.0 |
108.7 |
18.4 |
19.3 |
7.5 |
5 |
8.7 |
5.8 |
7.3 |
2.5 |
25.7 |
8.5 |
104.7 |
2.5 |
22.0 |
1.7 |
10 |
7.3 |
4.5 |
8.3 |
5.8 |
34.3 |
3.5 |
100.7 |
17.4 |
18.0 |
3.6 |
Positive control** |
7301.5 |
- |
445.0 |
- |
263.0 |
- |
450.5 |
- |
254.5 |
- |
*Solvent control = negative control: 100 µL DMSO
**Mutagens positive controls:
- 2.N.F. (0.001 mg/plate) in TA98 and TA1538 strains
- M.M.S. (0.1 mg/plate) in TA100 strain
- E.M.S. (10 mg/plate) in TA1535 strain
- 9 A.A. ( 0.05 mg/plate) in TA 1537 strain
Table 7.6.1/7:Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (S9 mix) in second test (pre-incubation method)
TFAS Concentration |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
TA 1538 |
|||||
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
|
0* |
7.7 |
2.0 |
10.3 |
3.5 |
44.0 |
3.6 |
117.0 |
8.6 |
29.3 |
5.7 |
0.1 |
9.7 |
3.2 |
8.0 |
2.6 |
38.0 |
6.5 |
136.7 |
1.1 |
29.0 |
4.6 |
0.5 |
10.0 |
4.0 |
8.7 |
2.9 |
43.0 |
9.8 |
125.3 |
13.0 |
29.7 |
5.7 |
1 |
11.3 |
1.1 |
9.7 |
2.5 |
41.3 |
2.9 |
125.7 |
11.0 |
25.3 |
1.5 |
5 |
10.0 |
2.6 |
8.3 |
3.2 |
44.3 |
5.8 |
120.0 |
9.8 |
22.3 |
3.0 |
10 |
7.7 |
3.0 |
7.7 |
1.1 |
46.3 |
5.1 |
111.3 |
17.4 |
22.0 |
4.6 |
Positive control** |
151.5 |
- |
188.0 |
- |
1510.0 |
- |
2241.5 |
- |
601.0 |
- |
*Solvent control = negative control: 100 µL DMSO
**Mutagens positive controls:
- 2.A. (0.002 mg/plate)
Table 1:Summary table ofChromosome Aberration Assay 1 and 3 without metabolic activation
Concentration (μg/mL) |
Time of Treatment / Sampling |
Relative Survival# |
Insolubility## |
Mean % aberrant cells### |
Chlorodifluoroacetic acid without metabolic activation (-S9) |
||||
Negative (solvent) control (A1/A3) |
3h/ 20h |
100 / 100 |
– |
0.75 |
5000μg/mL (A1/A3) |
3h/ 20h |
0 / 0 |
– |
NE |
4900μg/mL (A1) |
3h / 20h |
0 |
– |
NE |
4800μg/mL (A1) |
3h / 20h |
0 |
– |
NE |
4700μg/mL (A1) |
3h / 20h |
26 |
– |
NE |
4600μg/mL (A1) |
3h / 20h |
42 |
– |
NE |
4500μg/mL (A1/A3) |
3h / 20h |
38 / 33 |
– |
2.5 |
4400μg/mL (A1) |
3h / 20h |
55 |
– |
NE |
4300μg/mL (A1) |
3h / 20h |
52 |
– |
NE |
4200μg/mL (A1) |
3h / 20h |
55 |
– |
NE |
4100μg/mL (A1) |
3h / 20h |
55 |
– |
NE |
4000μg/mL (A1/A3) |
3h / 20h |
49 / 26 |
– |
1.0 |
3000μg/mL (A3) |
3h / 20h |
55 |
– |
2.0 |
2500μg/mL (A3) |
3h / 20h |
80 |
– |
NE |
2000μg/mL (A1/A3) |
3h / 20h |
72 / 96 |
– |
2.75 |
1500μg/mL (A3) |
3h / 20h |
65 |
– |
NE |
1000μg/mL (A1/A3) |
3h / 20h |
86 / 82 |
– |
0.75 |
500μg/mL (A3) |
3h / 20h |
93 |
– |
NE |
Positive control (A1/A3) |
3h/ 20h |
89 / 79 |
– |
3.5* |
Table 2:Summary table ofChromosome Aberration Assay 1 with metabolic activation
Concentration (μg/mL) |
Time of Treatment / Sampling |
Relative Survival# |
Insolubility## |
Mean % aberrant cells### |
Chlorodifluoroacetic acid with metabolic activation (+S9) |
||||
Negative (solvent) control |
3h / 20h |
100 |
– |
2.0 |
5000μg/mL |
3h / 20h |
0 |
– |
NE |
4900μg/mL |
3h / 20h |
0 |
– |
NE |
4800μg/mL |
3h / 20h |
0 |
– |
NE |
4700μg/mL |
3h / 20h |
2 |
– |
NE |
4600μg/mL |
3h / 20h |
25 |
– |
NE |
4500μg/mL |
3h / 20h |
27 |
– |
NE |
4400μg/mL |
3h / 20h |
37 |
– |
NE |
4300μg/mL |
3h / 20h |
41 |
– |
NE |
4200μg/mL |
3h / 20h |
38 |
– |
NE |
4100μg/mL |
3h / 20h |
37 |
– |
NE |
4000μg/mL |
3h / 20h |
47 |
– |
2.0 |
2000μg/mL |
3h / 20h |
76 |
– |
2.0 |
1000μg/mL |
3h / 20h |
81 |
– |
0.0 |
Positive control |
3h / 20h |
54 |
– |
85.7*** |
Table 3:Summary table ofChromosome Aberration Assay 2 without metabolic activation
Concentration (μg/mL) |
Time of Treatment / Sampling |
Relative Survival# |
Insolubility## |
Mean % aberrant cells### |
Chlorodifluoroacetic acid without metabolic activation (-S9) |
||||
Negative (solvent) control |
20h / 28h |
100 |
– |
0.0 |
5000μg/mL |
20h / 28h |
0 |
– |
NE |
4900μg/mL |
20h / 28h |
0 |
– |
NE |
4800μg/mL |
20h / 28h |
0 |
– |
NE |
4700μg/mL |
20h / 28h |
0 |
– |
NE |
4600μg/mL |
20h / 28h |
0 |
– |
NE |
4500μg/mL |
20h / 28h |
6 |
– |
NE |
4400μg/mL |
20h / 28h |
16 |
– |
NE |
4300μg/mL |
20h / 28h |
8 |
– |
NE |
4200μg/mL |
20h / 28h |
12 |
– |
NE |
4100μg/mL |
20h / 28h |
15 |
– |
NE |
4000μg/mL |
20h / 28h |
19 |
– |
1.5 |
2000μg/mL |
20h / 28h |
75 |
– |
1.5 |
1000μg/mL |
20h / 28h |
77 |
– |
0.0 |
500μg/mL |
20h / 28h |
88 |
– |
NE |
Positive control |
20h / 28h |
46 |
– |
46.2*** |
Negative (solvent) control: 1 (v/v) % Distilled water
Positive control (-S9): Ethyl methanesulfonate, 0.4 µL/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 7.6.1/1 Results of chromosome analysis – Experiment 1 (3 +17) with metabolic activation
|
Vehicle |
1000 µg/mL |
1200 µg/mL |
1360 µg/mL |
NQC |
|
Cytotoxicity |
no |
|
|
|
yes |
|
Cells scored |
201 |
202 |
202 |
200 |
100 |
|
Gaps |
0 |
0 |
0 |
0 |
|
|
Chromosome aberrations |
Deletions |
1 |
0 |
0 |
1 |
|
Exchange |
0 |
0 |
0 |
0 |
|
|
Chromatid aberrations |
Deletions |
1 |
1 |
1 |
1 |
|
Exchange |
0 |
1 |
0 |
0 |
|
|
Aberrant cell |
+ Gaps |
2 |
2 |
1 |
2 |
|
- Gaps |
2 |
2 |
1 |
2 |
|
|
Mitotic index(%) |
8.7 |
9.2 |
8.75 |
8.55 |
|
|
Mitotic index ± SD |
|
|
|
|
|
|
Numerical aberrations |
Polyploid |
0 |
1 |
1 |
0 |
0 |
Hyperdiploid |
1 |
0 |
1 |
0 |
0 |
|
Endoreduplicated |
0 |
1 |
0 |
0 |
0 |
|
Table 7.6.1/2 Results of chromosome analysis – Experiment 1 (3 +17) without metabolic activation
|
Vehicle |
1000 µg/mL |
1200 µg/mL |
1360 µg/mL |
NQC |
|
Cytotoxicity |
no |
|
|
|
yes |
|
Cells scored |
202 |
201 |
200 |
201 |
182 |
|
Gaps |
0 |
1 |
0 |
0 |
|
|
Chromosome aberrations |
Deletions |
2 |
1 |
1 |
1 |
|
Exchange |
0 |
0 |
0 |
0 |
|
|
Chromatid aberrations |
Deletions |
3 |
3 |
2 |
1 |
|
Exchange |
0 |
0 |
0 |
0 |
|
|
Aberrant cell |
+ Gaps |
5 |
5 |
3 |
2 |
|
- Gaps |
5 |
4 |
3 |
2 |
|
|
Mitotic index(% of control) |
8.6 |
10 |
9.95 |
10.3 |
|
|
Mitotic index ± SD |
|
|
|
|
|
|
Numerical aberrations |
Polyploid |
0 |
0 |
0 |
1 |
0 |
Hyperdiploid |
2 |
1 |
0 |
0 |
0 |
|
Endoreduplicated |
0 |
0 |
0 |
0 |
0 |
|
Table 7.6.1/3 Results of chromosome analysis – Experiment 2 (3 +17) with metabolic activation
|
Vehicle |
1050 µg/mL |
1200 µg/mL |
1360 µg/mL |
CPA |
|
Cytotoxicity |
no |
|
|
|
yes |
|
Cells scored |
201 |
202 |
202 |
201 |
67 |
|
Gaps |
2 |
0 |
1 |
0 |
|
|
Chromosome aberrations |
Deletions |
0 |
0 |
0 |
1 |
|
Exchange |
0 |
0 |
0 |
0 |
|
|
Chromatid aberrations |
Deletions |
3 |
0 |
1 |
2 |
|
Exchange |
0 |
0 |
0 |
0 |
|
|
Aberrant cell |
+ Gaps |
5 |
0 |
2 |
3 |
|
- Gaps |
3 |
0 |
1 |
3 |
|
|
Mitotic index(% of control) |
8.275 |
7.9 |
7.2 |
6.45 |
|
|
Mitotic index ± SD |
|
|
|
|
|
|
Numerical aberrations |
Polyploid |
1 |
0 |
1 |
1 |
0 |
Hyperdiploid |
0 |
1 |
0 |
0 |
0 |
|
Endoreduplicated |
0 |
1 |
1 |
0 |
0 |
|
Table 7.6.1/4 Results of chromosome analysis – Experiment 2 (20) without metabolic activation
|
Vehicle |
750 µg/mL |
1200 µg/mL |
1360 µg/mL |
CPA |
|
Cytotoxicity |
no |
|
|
|
yes |
|
Cells scored |
200 |
201 |
203 |
201 |
75 |
|
Gaps |
1 |
2 |
1 |
0 |
|
|
Chromosome aberrations |
Deletions |
2 |
0 |
0 |
1 |
|
Exchange |
0 |
0 |
0 |
2 |
|
|
Chromatid aberrations |
Deletions |
3 |
0 |
1 |
0 |
|
Exchange |
0 |
0 |
0 |
0 |
|
|
Aberrant cell |
+ Gaps |
6 |
2 |
2 |
3 |
|
- Gaps |
5 |
0 |
1 |
3 |
|
|
Mitotic index(% of control) |
8.25 |
7.15 |
5.35 |
5.4 |
|
|
Mitotic index ± SD |
|
|
|
|
|
|
Numerical aberrations |
Polyploid |
0 |
0 |
1 |
0 |
0 |
Hyperdiploid |
0 |
1 |
2 |
1 |
0 |
|
Endoreduplicated |
0 |
0 |
0 |
0 |
0 |
|
Table 7.6.1/1:Experiment 1 (3 hour treatment in the absence and presence of S-9)
Treatment (µg/mL)
|
- S-9 |
Treatment (µg/mL)
|
+S-9 |
||||
%RS |
MF§ |
%RS |
MF§ |
||||
0 |
100 |
1.27! |
|
0 |
100 |
3.29 |
|
200 |
129 |
4.02 |
NS |
200 |
90 |
3.20 |
NS |
400 |
144 |
3.66 |
NS |
400 |
97 |
3.21 |
NS |
600 |
107 |
2.15 |
NS |
600 |
105 |
4.03 |
NS |
800 |
117 |
2.17 |
NS |
800 |
86 |
3.65 |
NS |
1000 |
110 |
3.25 |
NS |
1000 |
86 |
2.87 |
NS |
1200 |
119 |
2.84 |
NS |
1200 |
90 |
3.47 |
NS |
1360 |
97 |
3.71 |
NS |
1360 |
84 |
1.65 |
NS |
Linear trend NS |
Linear trend NS |
||||||
NQO |
B[a]P |
||||||
0.1 |
90 |
24.82 |
|
2 |
74 |
14.42 |
|
0.15 |
69 |
46.58 |
|
3 |
34 |
23.58 |
|
§ = 6‑TG resistant mutants/106viable cells 7 days after treatment
%RS = Percent relative survival adjusted by post treatment cell counts
NS = Not significant
! = Based on one replicate only
Table 7.6.1/2:Experiment 2 (3 hour treatment in the absence and presence of S-9)
Treatment (µg/mL)
|
- S-9 |
Treatment (µg/mL)
|
+S-9 |
||||
%RS |
MF§ |
%RS |
MF§ |
||||
0 |
100 |
0.37 |
|
0 |
100 |
2.94 |
|
150 |
98 |
0.75 |
NS |
150 |
111 |
5.94 |
NS |
300 |
87 |
0.86 |
NS |
300 |
107 |
0.89 |
NS |
500 |
82 |
0.20 |
NS |
500 |
111 |
0.78 |
NS |
700 |
99 |
1.15 |
NS |
700 |
115 |
1.46 |
NS |
900 |
91 |
0.71 |
NS |
900 |
113 |
0.28 |
NS |
1100 |
76 |
1.19 |
NS |
1100 |
112 |
0.69 |
NS |
1360 |
80 |
0.78 |
NS |
1360 |
112 |
0.86 |
NS |
Linear trendNS |
Linear trendNS |
||||||
NQO |
B[a]P |
||||||
0.1 |
71 |
5.94 |
|
2 |
80 |
21.34 |
|
0.15 |
36 |
16.29 |
|
3 |
62 |
6.38 |
|
§ =6‑TG resistant mutants/106viable cells 7 days after treatment
%RS =Percent relative survival adjusted by post treatment cell counts
NS =Not significant
! =Based on one replicate only
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Chlorodifluoroacetic acid is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 400 mg/kg (the highest feasible concentration) under the experimental conditions.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 09 nov 2011 to 17 may 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP study, OECD 474 compliant
- 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 chlorodifluoroacetic acid and trifluoroacetic acid 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 this in vivo mutagenicity study with chlorodifluoroacetic acid was not performed as part of the EU REACH registration. For this reason no testing proposal for the in-vivo mutagenicity study was submitted.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Remarks:
- 4-8 october 2010
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Elevage Janvier (Route des Chènes Secs B.P. 4105 - 53940 LE GENEST-ST-ISLE, France)
- Age at study initiation: Approximately 7 weeks at the treatment
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- 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. Cages type II, polypropylene/polycarbonate (37 x 22.5 x 18 cm)
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M-Z+H H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH (D-59494, Soest, Germany), ad libitum.
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
- Acclimation period: At least 5 days under the same conditions as for the test
ENVIRONMENTAL CONDITIONS
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.3 – 24.9°C (preliminary experiment)
21.7 – 24.9°C (main test)
Relative humidity: 32 – 69 % (preliminary experiment)
36 – 67 % (main test)
Ventilation: 15 – 20 air exchanges/hour
IN-LIFE DATES: From 30 nov 2011 To 01 dec 2011 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: Phosphate buffer saline (x10)
- Justification for choice of solvent/vehicle: the most appropriate for the test and based on the solubility of CDFA - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Due to the highly acidic property (pH=2.1 in 5 % aqueous solution) and known corrosive effect of the test item, formulations were prepared in a PBS buffer solution, diluted such that the pH of the administered solution was not below pH 3.0. This pH adjustment had been considered not to adversely affect the determination of the test item ability to cause genotoxic effect in the experimental animals since the systemic form of the chemical in the blood was not changed by the gavage formulation.
A preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test. The preliminary toxicity test also determined whether there are large differences in toxicity between the sexes or not. Groups of two male and female mice were treated by oral gavage at two occasions with an approximately 3-hour interval in between at the dose levels of 400, 200, 100 and 50 mg/kg body weight/day. The concentration of the test item formulation was chosen to assure the same dosing volumes in mice for all dose levels (20 mL/kg body weight).
Animals were examined regularly for toxic signs and mortalities. The surviving mice were euthanized 48 hours after treatment. No bone marrow smears were prepared in the preliminary experiment. - Duration of treatment / exposure:
- 48 hours
- Frequency of treatment:
- twice
- Remarks:
- Doses / Concentrations:
0, 100, 200 and 400 mg/kg bw
Basis:
nominal conc. - No. of animals per sex per dose:
- 5 males per dose at 100 and 200 mg/kg bw
10 + 2 males at 400 mg/kg bw - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- - Positive control: cyclophosphamide
- Justification for choice of positive control(s): usually used by the lab, and recommended by the guideline
- Route of administration: gavage
- Doses / concentrations: 60 mg/kg bw (6 mg/mL) - Tissues and cell types examined:
- 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. Fewer than 2000 PCEs may be counted in the event of a clear positive effect.
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. - Details of tissue and slide preparation:
- PREPARATION OF BONE MARROW
asphyxiation with ascending doses of carbon dioxide. Deep anaesthesia was confirmed before making incision; death was confirmed by cervical dislocation or by cutting through major cervical blood vessels before discarding carcasses. 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.
TO BE COMPLETED - Evaluation criteria:
- EVALUATION OF THE RESULT:
The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups will be compared to the values found in the corresponding negative control group. Statistically analysis will be performed using Kruskall Wallis Non Parametric ANOVA test. Cytotoxicity will be expressed by the PCE/NCE ratio.
The test item will be considered to have shown genotoxic activity in this study if the following criteria are met:
- increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative control
- the increases are dose-related
- the increases are statistically significant.
The historical control range for this laboratory will also be considered when evaluating the biological significance of small increases.
The test item will be concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historic control values.
The Micronucleus Test is considered acceptable if it meets the following criteria:
- the frequencies of micronucleated polychromatic erythrocytes found in the negative and /or solvent controls falls within the range of historical laboratory control data.
- the positive control item should produce biologically relevant increases in the number of micronucleated polychromatic erythrocytes.
- Each treated and control group should include at least 5 analysable animals. - Statistics:
- Kruskal-Wallis test
- 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
The solubility limit of the test item in the selected vehicle was examined in a Preliminary Compatibility Test. Based on the observed results, 10 mg/mL formulation using 10X PBS as vehicle was used as the highest feasible concentration formulation in the preliminary toxicity test (higher concentration test item solutions had a pH value lower than 3.0). Taking the highest applicable treatment volume (20 mL/bw kg) and two treatments/day into account, the highest feasible concentration of the test item was 400 mg/body weight/day.
Therefore, groups of two male and female mice were treated at two occasions by oral gavage at the dose levels of 400, 200, 100 and 50 mg/kg bw/day in the Preliminary Toxicity Test.
Based on the results of the preliminary experiment, dose levels of 400, 200 and 100 mg/kg body weight/day were selected for the micronucleus test. Because the toxic effect of the test item was similar in both sexes in the preliminary toxicity test, the main experiment was performed using male mice only.
RESULTS OF DEFINITIVE STUDY
Groups of five male mice were treated with the test item at 400, 200 and 100 mg/kg body weight/day or with the vehicle (10X PBS) in the main experiment. All mice in the vehicle and test item groups were dosed by oral gavage at two occasions using a dose volume of 20 mL/kg body weight. Animals of the positive control group were treated by intraperitoneal injection with Cyclophosphamide at 60 mg/kg body weight/day at one occasion using a dose volume of 10 mL/kg body weight.
No marked effect of test item treatment on the body weight of the mice was observed in the main test.
No mortality or signs of systemic toxicity were observed during the study. The animals in each dose group, furthermore in the negative (vehicle) control and positive control groups were symptom-free during the whole observation period.
2000 polychromatic erythrocytes (PCEs) were scored per animal to assess 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 erythrocytes was determined for each animal by also counting mature erythrocytes (NCEs) until a total of 1000 PCEs+NCEs had been observed. Results are shown in Appendix 6.
Comparison of the vehicle control data and the high dose of the test item (400 mg/kg bw/day) at 24h using the Kruskal-Wallis test gave a value of H = 0.918. This is non-significant, giving a negative response. The average number of micronuclei in the high dose group at 48h was less than the negative control group; therefore statistical analysis was not necessary at that time point.
No indication of an increase in the number of micronucleated normochromatic erythrocytes was observed in the study.
- Statistical evaluation: Kruskal-Wallis test - Conclusions:
- No induction of micronuclei in bone marrow erythrocytes was observed following administration of Chlorodifluoroacetic acid to mice at up to and including 400 mg/kg bw/day (highest feasible concentration); thus, there was no evidence of any in vivo genotoxic activity of the test item under the conditions of this study.
- Executive summary:
The objective of the study (2012) was to determine whether Chlorodifluoroacetic acid test item caused genotoxic effects resulting in the formation of micronuclei in erythrocytes of treated mice in accordance with the OECD guideline “Mammalian Erythrocyte Micronucleus Test”, No. 474 (1997).
In the main test, groups of male mice were treated with the vehicle (10X PBS) or the test item at 400, 200 and 100 mg/kg body weight/day by oral gavage or the positive control item (Cyclophosphamide dissolved in physiological saline) at 60 mg/kg body weight/day administered by intraperitoneal injection. Five mice from each group were examined 24 hours after dosing, and a further five mice dosed with the vehicle or test item at 400 mg/kg body weight/day were examined 48 hours after dosing. Bone marrow smears were prepared on glass slides for each of the mice, stained, and scored. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells.
No marked effect on body weight was observed in the main test. No mortality or signs of systemic toxicity were observed during the study. The animals in each group were symptom-free during the whole observation period.
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. The positive control treatment caused a large, clearly positive response demonstrating the sensitivity of the test system.
In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Chlorodifluoroacetic acid to mice at up to and including 400 mg/kg bw/day (highest feasible concentration); thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.
Reference
Micronucleus Data
Mice sacrificed 24 hours after dosing
Treatment |
ID |
Animal |
MNPCE/ |
PCE/1000 PCE+NCE |
Group 1 |
8190 |
19 |
0 |
270 |
Negative (vehicle) control |
8202 |
31 |
1 |
205 |
(Phosphate buffered saline) |
8203 |
32 |
1 |
260 |
|
8207 |
36 |
1 |
291 |
|
8210 |
39 |
0 |
220 |
Mean: |
|
|
0.6 |
249.2 |
SD: |
|
|
0.548 |
35.72 |
Group 2 |
8173 |
2 |
1 |
350 |
Chlorodifluoroacetic acid |
8191 |
20 |
1 |
682 |
(100 mg/kg bw/day) |
8198 |
27 |
0 |
320 |
|
8204 |
33 |
1 |
430 |
|
8205 |
34 |
1 |
446 |
Mean: |
|
|
1.2 |
399.4 |
SD: |
|
|
1.095 |
38.33 |
Group 3 |
8174 |
3 |
0 |
300 |
Chlorodifluoroacetic acid |
8186 |
15 |
0 |
155 |
(200 mg/kg bw/day) |
8187 |
16 |
1 |
402 |
|
8189 |
18 |
1 |
393 |
|
8200 |
29 |
1 |
507 |
Mean: |
|
|
0.6 |
351.4 |
SD: |
|
|
0.548 |
132.02 |
Group 4 |
8175 |
4 |
3 |
363 |
Chlorodifluoroacetic acid |
8195 |
24 |
1 |
391 |
(400 mg/kg bw/day) |
8212 |
41 |
1 |
460 |
|
8213 |
42 |
0 |
410 |
|
8216 |
45 |
1 |
373 |
Mean: |
|
|
0.8 |
445.6 |
SD: |
|
|
0.447 |
142.34 |
Group 5 |
8188 |
17 |
31 |
330 |
Positive Control |
8194 |
23 |
40 |
442 |
(Cyclophosphamide, |
8196 |
25 |
27 |
381 |
60 mg/kg bw/day) |
8206 |
35 |
40 |
409 |
|
8215 |
44 |
37 |
310 |
Mean: |
|
|
35.0 |
374.4 |
SD: |
|
|
5.788 |
54.61 |
Mice sacrificed 48 hours after dosing
Treatment |
ID |
Animal |
MNPCE/ 2000 PCE |
PCE/1000 PCE+NCE |
Group 1 |
8177 |
6 |
2 |
310 |
Negative (vehicle) control |
8192 |
21 |
0 |
156 |
(Phosphate buffered saline) |
8185 |
14 |
0 |
232 |
|
8181 |
10 |
1 |
258 |
|
8208 |
37 |
0 |
210 |
Mean: |
|
|
0.6 |
233.2 |
SD: |
|
|
0.894 |
57.04 |
Group 4 |
8180 |
9 |
0 |
283 |
Chlorodifluoroacetic acid |
8182 |
11 |
0 |
278 |
(400 mg/kg bw/day) |
8184 |
13 |
0 |
211 |
|
8201 |
30 |
0 |
282 |
|
8211 |
40 |
0 |
322 |
Mean: |
|
|
0.0 |
275.2 |
SD: |
|
|
0.000 |
40.08 |
MNPCE: Number of Micronucleated Polychromatic Erythrocytes referring to counts of 2000 PCE.
PCE: Polychromatic Erythrocyte
NCE: Normochromatic Erythrocyte
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
No data is available on the reaction mass of chlorodifluoroacetic acid and trifluoroacetic acid. Experimental data are available on CDFA. For TFA no data are available but since TFA is a strong acid, testing with tthe neutral salt sodium trifluoroacetate was considered appropriate to avoid cytotoxic effects on bacteria due to the extreme acid pH of TFA (pH=0.45).
For chlorodifluoroacetic acid, negative results were observed in the following studies:
- Ames test, 2 studies (OECD 471).
- Chromosome aberration (OECD 473).
- Micronucleus in vivo (OECD 474).
For sodium trifluoroacetate, negative results were observed in the following studies:
- Ames test (OECD 471).
- Chromosome aberration (OECD 473).
- Mouse lymphoma assay (OECD 476).
Based on the results from these studies, the reaction mass is considered to be not genotoxic nor mutagenic.
Justification for classification or non-classification
Based on the all available negative results on each component, the reaction mass of chlorodifluoroacetic acid and trifluoroacetic acid is considered to be not genotoxic nor mutagenic, and thus is not classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
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