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Genetic toxicity: in vitro

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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:
08 Oct - 27 Oct 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Comparable to guideline study. Only one positive control substance was tested in the presence of metabolic activation and not in all strains. However, the metabolic activation system had been characterised and checked for quality and function by its manufacturer.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only one positive control substance was tested in the presence of metabolic activation and not in all strains. However, the metabolic activation system had been characterised and checked for quality and function by its manufacturer.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Only one positive control substance was tested in the presence of metabolic activation and not in all strains. However, the metabolic activation system had been characterised and checked for quality and function by its manufacturer.
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
446-630-3
EC Name:
-
Cas Number:
181587-01-9
Molecular formula:
C13H9Cl2F3N4OS
IUPAC Name:
5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethanesulfinyl)-1H-pyrazole-3-carbonitrile
Details on test material:
- Name of test material (as cited in study report): RPA 107382
- Analytical purity: 93%
- Lot/batch No.: CDR 9706

Method

Target gene:
his operon (S. typhimurium) and trp operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented mammalian liver post-mitochondrial fraction MolTox™ S-9 (Molecular Toxicology Incorporated, USA), prepared from the livers of male Sprague Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Initial toxicity range-finder experiment (only in TA100) and first experiment: 8, 40, 200, 1000 and 5000 µg/plate with or without metabolic activation
Second experiment: 156.25#, 312.5, 625, 1250, 2500 and 5000 µg/plate with or without metabolic activation
#this concentration was used for treatments in the presence of S9 only
Vehicle / solvent:
- Vehicle/solvent used: DMSO (0.1 mL for plate-incorporation treatments, 0.05 mL for preincubation treatments)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene (5 µg/plate, -S9, TA98); sodium azide (2 µg/plate, -S9, TA100 and TA 1535); 9-aminoacridine (50 µg/plate, -S9, TA1537); 4-nitroquinoline 1-oxide (2 µg/plate, -S9, WP2 uvrA); 2-aminoanthracene (+S9, 5-20 µg/plate, TA 98, TA 100, WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION 1: in agar (plate incorporation) for the initial toxicity range-finder test and first experiment

DURATION
- Exposure duration: 3 days

METHOD OF APPLICATION 2: preincubation followed by plate incorporation for the second experiment

DURATION
- Preincubation period: 1 h
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: triplicate each for treatments and positive controls and quintuplicate each for solvent controls in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn
Evaluation criteria:
The test article was considered to be mutagenic if the following criteria were met:
1) the assay was valid (mean negative control counts fell within the normal ranges of historical negative (solvent) controls; the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S9 preparation; no more than 5% of the plates were lost through contamination or some other unforeseen event.)
2) Dunnett's test gave a significant response (p < 0.01) and the data set(s) showed a significant dose correlation
3) the increases in revertant numbers in the positive controls were reproducible.

Statistics:
Mean values, fold increases over control and standard deviations were calculated. Statistical analysis included the determination of correlation coefficient, slope of best fit and significance. Statistical significance was indicated at *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 using Dunnett's test. The m-statistic was calculated to check that the data were Poisson-distributed.

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:
plate incorporation: at 5000 µg/plate; preincubation: at 2500 µg/plate and above (without S9), at 625 µg/plate and above (with S9)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in experiment 1 (plate incorporation), precipitation of the test substance was observed on all plates treated at 5000 µg/plate (maximum concentration). In experiment 2 (preincubation), precipitation of the test substance was observed on all plates treated at the higher test concentrations, both in the absence (at 1250 µg/plate and above) and in the presence (at 625 µg/plate and above) of S9.
- Positive controls in the presence of metabolic activation: only one positive control substance was tested in the presence of metabolic activation and not in all strains. However, each batch of the metabolic activation system was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufm-0-dealkylase activities). Therefore, these deviations were not considered to prejudice the validity of this study.

RANGE-FINDING/SCREENING STUDIES:
An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of the test substance at 8, 40, 200, 1000 and 5000 µg/plate, plus negative (solvent) and positive controls. Following these treatments, evidence of toxicity in the form of a thinning of the background bacterial lawn was observed at the maximum test concentration only in the presence of S9.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean numbers of spontaneous revertants in the negative (solvent) controls of the strains used were all within the normal historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment 1 (plate incorporation), evidence of toxicity was observed at the highest test concentration in the presence or absence of S9 in only a few strains (TA98 and TA1537) tested. In experiment 2 (preincubation), evidence of toxicity (in strains TA98, TA100, TA1537 and TA1535) was observed following several treatments at the higher test concentrations, both in the absence or in the presence of S9.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation)

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(Average of 3 (test substances) or 5 (solvent control) plates ± standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

WP2 uvr A

TA98

TA1537

0

108 ± 11

15 ± 3

15 ± 2

16 ± 3

7 ± 3

8

100 ± 5

13 ± 4

16 ± 7

16 ± 3

8 ± 2

40

112 ± 14

12 ± 3

15 ± 3

13 ± 4

11 ± 2

200

116 ± 13

11 ± 2

18 ± 4

14 ± 1

9 ± 5

1000

122 ± 21

12 ± 2

19 ± 5

19 ± 2

9 ± 2

5000

115 ± 6P

13 ± 4P

15 ± 4P

15 ± 3

6 ± 1P

Positive controls, -S9

Name

NaN3

NaN3

NQO

2NF

AAC

Concentrations (μg/plate)

2

2

2

5

50

Mean No. of colonies/plate (average of 3 ± SD)

661 ± 37

433 ± 66

1020 ± 60

126 ± 30

497 ± 12L

+

0

133 ± 13

21 ± 4

23 ± 2

29 ± 10

12 ± 1

+

8

137 ± 11

16 ± 2

27 ± 7

27 ± 12

13 ± 6

+

40

110 ± 14

17 ± 2

31 ± 3

27 ± 9

5 ± 2

+

200

126 ± 10

18 ± 8

19 ± 2

21 ± 11

10 ± 4

+

1000

130 ± 9

19 ± 6

19 ± 1

0

10 ± 4

+

5000

121 ± 17P,S

17 ± 2

21 ± 4

0

8 ± 1P,S

Positive controls, +S9

Name

AAN

-

AAN

AAN

-

Concentrations (μg/plate)

5

-

5

5

-

Mean No. of colonies/plate (average of 3 ± SD)

1518 ± 11

-

165 ± 34

301 ± 59

-

Table 2. Test results of experiment 2 (preincubation)

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(Average of 3 (test substances) or 5 (solvent control) plates ± standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

WP2 uvr A

TA98

TA1537

0

121 ± 13

20 ± 5

24 ± 3

26 ± 5

10 ± 3

312.5

118 ± 15

22 ± 5

26 ± 3

26 ± 10

6 ± 4

625

119 ± 1

18 ± 5

24 ± 4

26 ± 5

9 ± 2

1250

117 ± 4P

17 ± 7P

26 ± 4P

33 ± 2P

8 ± 2P

2500

107 ± 6P,S

20 ± 6P,S

27 ± 8P

29 ± 3P,S

5 ± 3P,S

5000

122 ± 4P,S

16 ± 1P,S

20 ± 3P

28 ± 4P,S

6 ± 1P,S

Positive controls, -S9

Name

NaN3

NaN3

NQO

2NF

AAC

Concentrations (μg/plate)

2

2

2

5

50

Mean No. of colonies/plate (average of 3 ± SD)

637 ± 76

462 ± 37

817 ± 52

850 ± 83

621 ± 127

+

0

151 ± 16

23 ± 3

24 ± 4

49 ± 8

15 ± 4

+

156.25

142 ± 10

19 ± 4

23 ± 5

39 ± 1

15 ± 4

+

312.5

141 ± 9

20 ± 4

30 ± 5

47 ± 2

16 ± 2

+

625

158 ± 8P,S

20 ± 3

25 ± 3P

41 ± 8P,S

13 ± 3P

+

1250

138 ± 6P,S

28 ± 9

23 ± 3P

32 ± 6P,S

9 ± 1P,S

+

2500

145 ± 8P,S

22 ± 11

26 ± 3P

38 ± 1P,S

10 ± 3P,S

+

5000

145 ± 2P,S

17 ± 4

23 ± 4P

36 ± 3P,S

11 ± 1P,S

Positive controls, +S9

Name

-

-

AAN

AAN

-

Concentrations (μg/plate)

-

-

10 + 20

5

-

Mean No. of colonies/plate (average of 3 ± SD)

-

-

10: 30 ± 10 20: 40 ± 6

1651 ± 213

-

NaN3 = sodium azide                   

NQO = 4-nitroquinoline 1-oxide                             

2NF = 2-nitrofluorene                  

AAC = 9-aminoacridine                

AAN = 2-aminoanthracene                       

P = Precipitate                 

S = Slight thinning of background bacterial lawn                             

L = Plate lost

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative