2-Pyridinesulfonamide, 3-(2,2,2-trifluoroethoxy)-, sodium salt (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted test guidance and good laboratory practices guidance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

sodium 2-(azanidylsulfonyl)-3-(2,2,2-trifluoroethoxy)pyridine

EC Number:

700-926-1

Cas Number:

227605-94-9

Molecular formula:

C7H6F3N2NaO3S

IUPAC Name:

sodium 2-(azanidylsulfonyl)-3-(2,2,2-trifluoroethoxy)pyridine

Constituent 2

Reference substance name:

Sodium {[3-(2,2,2-trifluoroethoxy)-2-pyridinyl]sulfonyl}azanide

IUPAC Name:

Sodium {[3-(2,2,2-trifluoroethoxy)-2-pyridinyl]sulfonyl}azanide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): CA 3105 A (intermediate of CGA 362622)
- Substance type: Organic mono constituent substance
- Physical state: White powder
- Analytical purity: 93.9%
- Lot/batch No.: EZ001002
- Expiration date of the lot/batch: July 2002 (reanalysis date)
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Method

Target gene:

hisitidine (Salmonella)
tryptophan (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

-Type and identity of media:
Liquid nutrient broth medium (NB-medium)
Standard plate incorporation assay: 0.1 mL of the overnight cultures were mixed with 2 mL of top agar, either 0.5 mLof 100 mM sodium phosphate buffer ( experiments without activation) or 0.5 mL of the activation mixture (experiments with activation) and 0.1 mL of a solution of the test item, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes.
Preincubation assay: 0.1 mL of the overnight cultures were mixed with 0.5 mL of the activation mixture ( experiments with activation) and 0.1 mL of a solution of the test item, 0.1 mL of the positive control or 0.1 mL of the solvent as a negative control and incubated for 30 min. at 37°C.
Thereafter 2 mL of top agar were added to the mixtures and they were poured on minimal agar in Petri dishes.
Each Petri dish contained about 20.0 mL of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCI. In the experiment with Salmonella the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM d-biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5 mM L-tryptophan dissolved in water.
-Properly maintained: yes
-Periodically checked for Mycoplasma contamination: Not provided.
-Periodically checked for karyotype stability: yes. The characteristics of the strains were checked every two to six months. Histidine-auxotrophy of the Salmonella strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene was demonstrated by the sensitivity for UV -light. The Salmonella strains containing the R-factor (TA 98 and TA I 00) were additionally checked for ampicillin resistance. The tryptophan-auxotrophy of E. coli WP2 uvrA was demonstrated by the requirement for tryptophan. The absence of the uvrA gene was demonstrated by the sensitivity of the strain for UV-light.
Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).
-Periodically”cleansed” against high spontaneous background: Not provided.

Species / strain 2

Details on mammalian cell type (if applicable):

-Type and identity of media:
Liquid nutrient broth medium (NB-medium)
Standard plate incorporation assay: 0.1 mL of the overnight cultures were mixed with 2 mL of top agar, either 0.5 mLof 100 mM sodium phosphate buffer ( experiments without activation) or 0.5 mL of the activation mixture (experiments with activation) and 0.1 mL of a solution of the test item, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes.
Preincubation assay: 0.1 mL of the overnight cultures were mixed with 0.5 mL of the activation mixture ( experiments with activation) and 0.1 mL of a solution of the test item, 0.1 mL of the positive control or 0.1 mL of the solvent as a negative control and incubated for 30 min. at 37°C.
Thereafter 2 mL of top agar were added to the mixtures and they were poured on minimal agar in Petri dishes.
Each Petri dish contained about 20.0 mL of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCI. In the experiment with Salmonella the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM d-biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5 mM L-tryptophan dissolved in water.
-Properly maintained: yes
-Periodically checked for Mycoplasma contamination: Not provided.
-Periodically checked for karyotype stability: yes. The characteristics of the strains were checked every two to six months. Histidine-auxotrophy of the Salmonella strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene was demonstrated by the sensitivity for UV -light. The Salmonella strains containing the R-factor (TA 98 and TA I 00) were additionally checked for ampicillin resistance. The tryptophan-auxotrophy of E. coli WP2 uvrA was demonstrated by the requirement for tryptophan. The absence of the uvrA gene was demonstrated by the sensitivity of the strain for UV-light.
Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).
-Periodically”cleansed” against high spontaneous background: Not provided.

Metabolic activation:

with and without

Metabolic activation system:

S9 (rat liver)

Test concentrations with justification for top dose:

Range finding test: 20.6, 61.8, 185.2, 555.6, 1666.7 and 5000.0 µg/plate
Mutagenicity test: 312.5, 625.0, 1250.0, 2500.0 and 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: bidistiled water (made in-house)
- Justification for choice of solvent/vehicle: CA 3105 A was dissolved in bidistilled water at room temperature. The test item was soluble up
to the concentration of 50 mg/mL. Lower concentrations of the test item were obtained by serial dilution of the stock solution with bidistilled water. No precipitates or aggregates were noted.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

+S9: Salmonella typhimurium TA 100, TA 102, TA 98, TA 1537 and E.coli WP2uvrA

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9: Salmonella typhimurium TA 1535

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-S9: Salmonella typhimurium TA 100, TA 1535

Controls 4

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline

Remarks:

-S9: E. coi WP2uvrA

Controls 5

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

-S9: Salmonella typhimurium TA 102

Controls 6

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

-S9: Salmonella typhimurium TA98

Controls 7

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

-S9: Salmonella typhimurium TA 1537

Details on test system and experimental conditions:

METHOD OF APPLICATION:
-in agar (plate incorporation): Original mutagenicity test with and without S9 and Confirmatory test without S9
-preincubation: Confirmatory test with S9


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 h
- Expression time (cells in growth medium): /
- Selection time (if incubation with a selection agent): /
- Fixation time (start of exposure up to fixation or harvest of cells): /

SELECTION AGENT (mutation assays): histidine (Salmonella), tryptophan (E.coli)

NUMBER OF REPLICATIONS: 3 (mutagenicity tests); 1 (range finding test)

NUMBER OF CELLS EVALUATED: /

DETERMINATION OF CYTOTOXICITY
- Method: other: background lawn

OTHER: OTHER: Colony counting and scoring of the plates
Colonies were counted electronically using an Accucount 1000 (Biologics, Gainsville, Virginia, USA), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have interfered with automating counting. The results were sent on line to a computer. The operator checked them on a random basis. Observations indicating precipitates of the test item in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated.

Evaluation criteria:

Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgment of the Study Director.

Criteria for a positive response
The test item will be considered to be positive in the test system if one or both of the following conditions are met:
• At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: TA 98, TA 1535, TA 1537, E. coli WP2 uvrA.
• A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strains TA 100 or T A 102.
Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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 applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES: Six concentrations of CA 3105 A (Intermediate of CGA 362622) ranging from 20.6 to 5000.0 µg/plate were tested with strain Salmonella typhimurium TA 100 and strain Escherichia coli WP2uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. The numbers of revertant colonies were not reduced. Normal background growth was observed with both strains. The test item did not precipitate on the surface of the agar plates.
From the results obtained, the highest concentrations suitable for the original mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: yes (Arithmetic Mean and Standard Deviation (SD) of colony counts in N experiments reported in the period of January 01, 1999 to December 31, 2000 and acceptable ranges for mean colony counts of spontaneous revertants.)

ADDITIONAL INFORMATION ON CYTOTOXICITY: No.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the results of these experiments and on standard evaluation criteria, it is concluded that CA 3105 A (Intermediate of CGA 362622) and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Executive summary:

CA 3105 A (Intermediate of CGA 362622), identified as white powder, purity 93.9%, batch no.EZ001002, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The test item was dissolved in bidistilled water and tested at five concentrations in the range of 312.5 to 5000.0 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 312.5 to 5000.0 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

The original experiment with and without metabolic activation and the confirmatory experiment without activation were performed as standard plate incorporation assay. The confirmatory experiment with metabolic activation was carried out as preincubation assay.

In all experiments, performed with and without metabolic activation, none of the tested concentrations of CA 3105 A led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control.

 

Based on the results of these experiments and on standard evaluation criteria, it is concluded that CA 3105 A (Intermediate of CGA 362622) and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.