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Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
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

Constituent 1
Chemical structure
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 / strainopen allclose all
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 / 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 / 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 resultsopen allclose all
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
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
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.

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