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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Remarks:
No deviations ocurred that impacted the integrity of the study.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1,2,2,3,3,4,4,4-nonafluoro-N-methylbutane-1-sulfonamide
EC Number:
614-396-3
Cas Number:
68298-12-4
Molecular formula:
C5H4F9NO2S
IUPAC Name:
1,1,2,2,3,3,4,4,4-nonafluoro-N-methylbutane-1-sulfonamide
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
3M Company, Lot 3
- Expiration date of the lot/batch:
06 August, 2002
- Purity test date:
30 January, 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature in the dark.
- Stability under storage conditions:
Stable
- Stability under test conditions:
Stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test article was suspended in DMSO
- Final preparation of a solid:
Suspended in DMSO

FORM AS APPLIED IN THE TEST: Suspended in DMSO

Method

Target gene:
Tryptophan and hisitdine operons
Species / strain
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:
Type and composition of metabolic activation system:
- source of S9 : Aroclor-1254 induced rat liver S9-mix
- method of preparation of S9 mix: Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany.
- concentration or volume of S9 mix and S9 in the final culture medium: 5% v/v in the first experiment and 10% v/v in the second experiment.
- quality controls of S9: Before use, all Sg-batches were characterized with the metabolic activation requiring positive control; benzo[a]pyrene (Sigma) in tester strain TA98 at the concentration of 5 ug/plate
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 3330, 5000 ug/plate. The top dose is the limit dose per OECD 471.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Test article and test system compatibility.

- Justification for percentage of solvent in the final culture medium: No data
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycine, 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^9 cells/mL
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: None
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): Colonies were counted following the 48 hour exposure.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 hours.
- Selection time (if incubation with a selective agent): 48 hours (Histidine and tryptophan-minimal agar)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: Histidine and tryptophan-minimal agar.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: background growth inhibition


METHODS FOR MEASUREMENTS OF GENOTOXICIY : See "Evaluation Criteria" section.

Rationale for test conditions:
Per OECD 471.
Evaluation criteria:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicit4y was observed at 5000 ug/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 3330 ug/plate and greater.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 5000 ug/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 3330 ug/plate and greater.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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:
cytotoxicity
Remarks:
Cytotoxicity was observed at 5000 ug/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: None
- Data on osmolality: None
- Possibility of evaporation from medium: The test article is a solid suspended in a vehicle. Evaporation is not expected/possible
- Water solubility: No data
- Precipitation and time of the determination: No precipitation was observed.

RANGE-FINDING/SCREENING STUDIES (if applicable): The range-finding study was included as the first portion of the first mutation experiment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : No dose-related, two-fold, increase in the number of revertants was observed in two independently repeated experiments.
- Statistical analysis; p-value if any - No data

Ames test:
- Signs of toxicity : Toxicity (reduction of the bacterial background lawn and reduction in the number of revertants) was observed inall stains at 3330 and/or 5000 ug/plate in the presence and absence of S9-mix.
- Individual plate counts : See attached results tables for summary.
- Mean number of revertant colonies per plate and standard deviation : See attached results tables for summary.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: See attached table.
- Negative (solvent/vehicle) historical control data: See attached table.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test article is not mutagenic in the Bacterial Reverse Mutation Assay in the presence or absence of metabolic activation.
Executive summary:

 The mutagenic potential of the test article (off white solid, Lot 3) was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA in the presence and absence of metabolic activation (S9-mix:Aroclor-1254 induced rat liver). This study was performed in compliance with OECD GLP. The study method was based on OECD 471 (1997) and EEC Directive 67/548/EEC B.13-14 (2000). The test article was prepared in dimethyl sulfoxide just prior to administration to the cells. In the dose range-finding test, the test article was tested at up to 5000 ug/plate in TA100 and WP2uvrA in the presence and absence of S9-mix. In the mutation assays, the test article was tested at up to 5000 ug/plate in each strain in the presence and absence of S9-mix. Strain-specific positive controls were tested in parallel. Each treatment was performed in triplicate. Toxicity (reduction of the bacterial background lawn and reduction in the number of revertants) was observed in all stains at 3330 and/or 5000 ug/plate in the presence and absence of S9-mix. No reproducible, dose-related increase in the number of revertant colonies was observed in any strain at any dose in the presence or absence of S9-mix. All criteria for a valid study were met. Under the conditions of this study, the test article is not mutagenic in the Bacterial Reverse Mutation Assay in the presence or absence of metabolic activation.