2-Pyridinesulfonamide, N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(2,2,2-trifluoroethoxy)-, sodium salt (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 July 1997 to 8 October 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to GLP and in line with standardised guidelines OECD 471, EU Method B.14 and EPA OPPTS 798.5265 with no deviations thought to impact on the reliability of the presented results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MITI (The Ministry of International Trade and Industry), Japan 62 Notification of Basic Industries Bureau No. 1014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

sodium 4,6-dimethoxy-N-({[3-(2,2,2-trifluoroethoxy)pyridin-2-yl]sulfonyl}carbamoyl)pyrimidin-2-aminide

EC Number:

688-332-8

Cas Number:

199119-58-9

Molecular formula:

C14H13F3N5O6SNa

IUPAC Name:

sodium 4,6-dimethoxy-N-({[3-(2,2,2-trifluoroethoxy)pyridin-2-yl]sulfonyl}carbamoyl)pyrimidin-2-aminide

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: solid (powder)
- Stability under test conditions: Stable
- Storage condition of test material: room temperature

Method

Target gene:

S. typhimurium: Histidine synthesis

E. coli: Tryptophan synthesis

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient broth medium
- Periodically checked for genotype stability: Yes. Histidine auxotrophy 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. TA 98 and TA 100 containing the R-factor were additionally checked for ampicillin resistance.
- Periodically "cleansed" against high spontaneous background: Yes. All strains were tested for amino acid requirement.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient broth medium
- Periodically checked for genotype stability: Yes. Histidine auxotrophy 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.
- Periodically "cleansed" against high spontaneous background: Yes. All strains were tested for amino acid requirement.

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient broth medium
- Periodically checked for genotype stability: Yes. Tryptophan auxotrophy was demonstrated by the requirement for tryptophan. The absence of the uvrA gene was demonstrated by the sensitivity of the strain for UV-light.
- Periodically "cleansed" against high spontaneous background: Yes. All strains were tested for amino acid requirement.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0.08, 0.25, 0.76, 2.29, 6.86 µg/plate (S. typhimurium) both in the presence and absence of metabolic activation
61.73, 185.19, 555.56, 1666.67, 5000.0 µg/plate (E. coli WP2uvrA) both in the presence and absence of metabolic activation
1st confirmatory assays: as above
2nd confirmatory assays:
- TA 1535: 0.76, 2.29, 6.86, 20.57, 61.70 µg/plate both in the presence and absence of metabolic activation
- TA 102: 0.01, 0.03, 0.09, 0.25, 0.76 µg/plate both in the presence and absence of metabolic activation
- TA 98: 0.76, 2.29, 6.86, 20.57, 61.70 µg/plate in the absence of metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

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

2-nitrofluorene

sodium azide

cyclophosphamide

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar - plate incorporation (original experiment)
- preincubation assay (first and second confirmatory experiments)

DURATION
- Exposure duration: Plates were incubated inverted at 37 °C for 48 hours in the dark (plate incorporation). Plates were incubated at 37 ºC for 30 minutes (preincubation assay)

NUMBER OF REPLICATIONS: Test concentrations were performed in triplicate

- EVALUATION PROCEDURE: Following the total incubation period the plates were examined for evidence that the test was valid: i.e. there was a background lawn on the solvent control plates and that the positive controls had responded as expected. All plates were counted using an electronic colony counter.

Evaluation criteria:

Test data from individual experiments are considered valid if:
a) the concurrent solvent control data are acceptable
b) the positive control data show acceptable increases

A positive response in a (valid) individual experiment is achieved when one or both of the following are met:
a) at least a reproducible doubling of the mean number of revertants per plate above that of the solvent control at any concentration for one or more of the following strains: TA 98, TA 1535, TA 1537, E coli WP2 uvrA ;
b) a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control be at least a factor of 1.5 for strains TA 100 or TA102.
Generally a concentration-related effect should be demonstrable.

Statistics:

Statistical analyses were not performed.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material was found to have a growth inhibiting effect.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material was found to have a growth inhibiting effect.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test compound did not precipitate at and concentration tested.

RANGE-FINDING/SCREENING STUDIES:
A range finding test was carried out with strains S. Typhimurium TA 100 and E. Coli WP2 uvrA with and without metabolic activation at six concentrations of the test material and one solvent control. The highest concentration applied was 5000.0 µg/plate. The five lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37 ± 12.5 ºC in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test material concentration and solvent control was used.However, since considerable growth inhibiting effects of the test material were seen in strain TA 100, an additional experiment with this strain with and without metabolic activation was performed using concentrations of 0.08 – 20.6 µg/plate. No background growth was observed with strain TA 100 at the concentrations of 20.6 to 5000.0 µg/plate. The numbers of revertant colonies were reduced at the concentrations of 6.87 to 5000.0 µg/plate. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate (E. Coli WP2 uvrA) and 6.86 µg/plate (S. Typhimurium) with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the study were found to be comparable with the historical control.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Due to a growth inhibiting effect, no background lawn could be observed in the experiments with metabolic activation on strain TA 100 (6.86 µg/plate), TA 1535 (61.7 µg/plate) and TA 102 (2.29 and 6.86 µg/plate). In the experimetns without activation the background lawn was invisible on strains TA 1535 (61.7 µg/plate) and TA 102 (2.29 and 6.86 µg/plate). Also the number of revertant colonies was reduced int he experiments with metabolic activation on strains TA 100, TA 98, TA 1537 (6.86 µg/plate), TA 1535 (20.57 and 61.7 µg/plate) and TA 102 (0.76 to 6.86 µg/plate). In the experiment without metabolic activation a reduction in the number of revertant colonies occurres on strains TA 100 (6.86 µg/plate), TA 1535 (20.57 and 61.7 µg/plate), TA 102 (0.76 to 6.86 µg/plate), TA 98 (61.7 µg/plate), TA 1537 (2.29 and 6.86 µg/plate) and E.coli WP2uvrA (5000.0 µg/plate).

Any other information on results incl. tables

Table 2: Summary of Original Mutagenicity Experiment

Strain	Treatment	Mean counts (+ S9)	Mean counts (-S9)
TA 100	solvent control	108.33	100.67
	0.08 µg/plate	102.33	100.00
	0.25 µg/plate	112.33	90.00
	0.76 µg/plate	101.67	94.00
	2.29 µg/plate	99.67	77.67
	6.86 µg/plate	19.33	15.67
	positive control	1491.67	1097.67
WP2 uvrA	solvent control	21.67	16.33
	61.73 µg/plate	17.67	18.67
	185.19 µg/plate	21.00	19.00
	555.56 µg/plate	20.67	17.67
	1666.67 µg/plate	18.33	18.33
	5000.0 µg/plate	17.33	17.67
	positive control	1077.67	605.67
TA 1537	solvent control	7.67	6.00
	0.08 µg/plate	8.67	6.33
	0.25 µg/plate	7.33	5.67
	0.76 µg/plate	8.33	6.67
	2.29 µg/plate	4.33	3.00
	6.86 µg/plate	3.67	1.67
	positive control	200.67	1349.67
TA 1535	solvent control	18.33	12.00
	0.08 µg/plate	17.00	10.00
	0.25 µg/plate	15.00	14.00
	0.76 µg/plate	14.00	13.33
	2.29 µg/plate	14.67	10.67
	6.86 µg/plate	15.00	8.33
	positive control	344.00	699.33
TA 98	solvent control	37.33	29.67
	0.08 µg/plate	37.33	25.33
	0.25 µg/plate	37.33	24.67
	0.76 µg/plate	39.67	29.67
	2.29 µg/plate	34.67	27.00
	6.86 µg/plate	33.67	21.33
	positive control	755.67	297.67
TA 102	solvent control	252.33	253.67
	0.08 µg/plate	229.00	239.00
	0.25 µg/plate	182.67	168.67
	0.76 µg/plate	61.67	15.67
	2.29 µg/plate	1.33	10.00
	6.86 µg/plate	0.00	1.33
	positive control	2358.33	1083.00

 

Table 3: Summary of 1st Confirmatory Experiment

Strain	Treatment	Mean counts (+ S9)	Mean counts (-S9)
TA 100	solvent control	136.33	136.00
	0.08 µg/plate	135.33	158.00
	0.25 µg/plate	162.00	143.00
	0.76 µg/plate	97.00	164.33
	2.29 µg/plate	138.00	149.33
	6.86 µg/plate	0.00	156.67
	positive control	1505.33	1158.00
WP2 uvrA	solvent control	24.00	26.33
	61.73 µg/plate	19.00	16.33
	185.19 µg/plate	17.33	15.33
	555.56 µg/plate	17.67	21.33
	1666.67 µg/plate	19.67	18.33
	5000.0 µg/plate	20.67	11.33
	positive control	495.00	447.67
TA 1537	solvent control	10.33	11.33
	0.08 µg/plate	8.33	8.00
	0.25 µg/plate	8.67	7.67
	0.76 µg/plate	8.67	7.00
	2.29 µg/plate	13.67	6.67
	6.86 µg/plate	2.33	7.00
	positive control	269.67	1109.00
TA 1535	solvent control	14.33	15.33
	0.08 µg/plate	16.67	18.67
	0.25 µg/plate	16.00	16.67
	0.76 µg/plate	22.67	16.00
	2.29 µg/plate	19.67	18.67
	6.86 µg/plate	10.00	19.33
	positive control	413.33	765.33
TA 98	solvent control	29.33	18.33
	0.08 µg/plate	28.00	18.67
	0.25 µg/plate	29.33	16.67
	0.76 µg/plate	26.33	13.00
	2.29 µg/plate	28.33	22.00
	6.86 µg/plate	14.67	14.33
	positive control	1075.67	300.33
TA 102	solvent control	264.33	271.33
	0.08 µg/plate	242.00	237.33
	0.25 µg/plate	221.33	172.33
	0.76 µg/plate	110.33	74.33
	2.29 µg/plate	41.00	21.00
	6.86 µg/plate	0.00	16.33
	positive control	1635.00	1296.33

 

Table 4: Summary of 2nd Confirmatory Experiment

Strain	Treatment	Mean counts (+ S9)	Mean counts (-S9)
TA 1535	solvent control	14.67	10.00
	0.76 µg/plate	12.00	12.00
	2.29 µg/plate	16.00	9.33
	6.86 µg/plate	12.67	10.00
	20.57 µg/plate	5.33	3.33
	61.70 µg/plate	0.00	0.00
	positive control	287.67	589.67
TA 102	solvent control	339.67	306.33
	0.01 µg/plate	330.33	275.00
	0.03 µg/plate	318.00	275.00
	0.09 µg/plate	243.33	214.33
	0.25 µg/plate	227.33	177.67
	0.76 µg/plate	96.33	32.00
	positive control	978.00	1220.67
TA 98	solvent control		12.00
	0.76 µg/plate		16.67
	2.29 µg/plate		13.33
	6.86 µg/plate		6.67
	20.57 µg/plate		8.00
	61.70 µg/plate		2.00
	positive control		275.67

 

Applicant's summary and conclusion

Conclusions:

Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 , TA 100 and TA 102 and E. coli strain WP2 uvrA both in the presence and absence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines OECD 471, EU Method B.14 and EPA OPPTS 798.5265. Five strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) and one Escherichia coli strain (WP2uvrA) were treated in the presence and absence of a rat liver derived metabolic activation system (S9 mix). The first confirmatory experiment was carried out with and without metabolic activation. Since marked differences in toxicity occurred with the strains used, a second confirmatory experiment was performed with and without metabolic activation on strains TA 1535 and TA 102. Strain TA 98 was tested without activation. 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 first and second confirmatory experiments with metabolic activation were carried out as preincubation assay.

 

In both experiments, performed with and without metabolic activation, none of the tested concentrations of test material led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the solvent control.