Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
15 March 2002 - 29 March 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

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

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:
other: EPA 712-C-98-247
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: CPMP/ICH/141/95
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: CPMP/ICH/174/95
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5-dimethylpyrazole
EC Number:
200-657-5
EC Name:
3,5-dimethylpyrazole
Cas Number:
67-51-6
Molecular formula:
C5H8N2
IUPAC Name:
3,5-dimethyl-1H-pyrazole
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Physical state: White powder
- Storage condition of test material: In the dark at ambient temperature

Method

Target gene:
his G 46 in TA 1535 and TA 100,
his C 3076 in TA 1537,
his D 3052 in TA 1538 and TA 98,
trpE locus in E. coli.

his G 46 is a mis-sense mutation which is reverted to prototrophy by a variety of mutagens that cause base-pair substitutions

his C 3076 contains a frameshift mutation which appears to have added a GC base pair. This mutation is reverted by 9-aminoacridine, ICR-191 and epoxides of polycyclic hydrocarbons.

his D 3052 also contains a frameshift mutation which is reverted by the deletion of 2 base-pairs, CG GC. It is readily reverted by aromatic amines and derivatives.

E.col WP2uvrA contains an ochre mutation at the trpE locus and can be mutated to tryptophan independence either by a base-pair reversion of an A-T base-pair at the tprE locus, or, more likely, by a base-pair substitution within a number of transfer RNA loci elsewhere in the chromome.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media: Oxoid Nutrient Broth No. 2 (25 g per litre).
- Properly maintained: yes
- Samples of each strain were grown by culturing for 16 hrs at 37 ºC in nutrient broth. Cultures were kept for up to 4 days at +4 ºC to allow relevant checks to be performed but fresh cultures were used for the experiment.
Additional strain / cell type characteristics:
other: rfa, uvrB, pKM101
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 mix
Test concentrations with justification for top dose:
- 17, 50, 167, 500, 1667 and 5000 µg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Two mutation assays were carried out; one with direct plate incorporation and the second with preincubation.

DURATION
- Direct plate method: Plates were prepared by mixing 0.5 mL of S9 mix or 0.05 M phosphate buffer and 0.1 mL of bacteria to 2 mL of soft agar, 0.1 mL aliquots of the solvent (DMSO) or test material were added last. Once mixed, the cooling agar was then poured onto minimal medium plates. These plates contained 25 mL of 1.5% purified agar, in Vogel-Bonner Medium E with 2% glucose. Once set the agar plates were inverted and incubated at 37º C.
- Preincubation period: Preparation began with adding 0.5 mL volumes of either S9 mix or 0.05 M phosphate buffer to sterile tubes, followed by 0.1 mL of bacteria and finally 0.1 mL of either the test solution or solvent (DMSO). Tubes were then sealed and placed in a shaking incubator at 37 ºC for 20 minutes. After which 2 mL of soft agar was added. The cooling contents were then mixed and poured into agar plates, as above. Once set the agar plates were inverted and incubated at 37 °C.
- Exposure duration: Plates were incubated for 2 or 3 days.

NUMBER OF REPLICATIONS: All concentrations were performed with and without S9 mix in triplicate for both tests.

COLONY EVALUATION: Colonies of ≥ 0.1 mm in diameter were counted.

TOXICITY TEST
- Strain: TA 100
- Concentrations: 17, 50, 167, 500, 1667 and 5000 µg per plate.
- Method: One plate was prepared for each concentration in the presence and absence of the S9 mix.

OTHER EXAMINATIONS
Quality Control: Each strain was tested for its resistance to amplicillin (indicating pKM101) and its sensitivity to ultraviolet light and crystal violet (indicating persistence of the uvrB and rfa mutations).
Evaluation criteria:
EVALUATION CRITERIA
A positive response was recorded if the following criteria were met:
1) For S. typhimurium strains TA 1353, TA 1537, and TA 98 and for E. coli at least doubling of the mean concurrent vehicle control values at some concentration of the test material. For S. typhimurium stain TA 100, a 1.5-fold increase over the control value was considered significant. If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases, a minimum count of 20 was required before a significant mutagenic response was registered.
2) A dose related response, although at high dose levels this relationship could be inverted because of, for example (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
3) A reproducible effect in independent tests.

ACCEPTABILITY
The test was considered acceptable if the following criteria were met:
1) The bacteria demonstrated their typical response to crystal violet, ampicillin and U.V. light.
2) At least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and E. coli WP2uvrA 1-60.
3) On at least 2 of the positive control plates, there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5.
4) No toxicity or contamination was observed in at least 4 dose levels.
5) In cases where a mutagenic response was observed, no more than one dose level was discarded before the dose that gave the highest significant mean colony number.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
other: All results were valid with the exception of the counts obtained for TA 100 with 9-aminoacridine. The mutant counts were higher than those reported in the historical data. However this was not thought to have affected the integrity of the study results.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precippitation was observed at any of the concentrations tested.

QUALITY CONTROL:
All strains were sensitive to crystal violet, whereas only the plasmid-containing stains, TA 98 and TA 100, were resistant to ampicillin. The strains were also tested for sensitivity to U.V light emitted over a period of 5-10 s from a lamp set at 254 nm. Increased sensitivity to U.V. light was demonstrated. These results are consistent with the known properties of these bacteria.

TOXICITY TEST:
No toxicity to bacteria was observed and no precipitation of the test material occurred in either the presence or absence of S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Direct Plate Method: Mean Number of Revertant Colonies in the Presence and Absence of S9 Mix

Substance

Concentrations ( µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

In the Presence of S9

DMSO

100 µL

22 ± 5

17 ± 6

25 ± 8

117 ± 8

9 ± 0

Test Material

17

21 ± 1

22 ± 1

22± 6

115 ± 15

11 ± 5

50

22 ± 1

15 ± 3

20 ± 2

116 ± 11

9 ± 3

167

18 ± 3

16 ± 4

24 ± 3

111 ± 6

14 ± 2

500

18 ± 5

19 ± 4

21 ± 7

118 ± 3

10 ± 2

1667

20 ± 2

19 ± 3

26 ± 6

115 ± 9

16 ± 3

5000

18 ± 6

15 ± 8

25 ± 2

117 ± 8

13 ± 2

In the Absence of S9

DMSO

100 µL

24 ± 8

14 ± 3

11 ± 4

110 ± 6

12 ± 6

Test Material

17

24 ± 11

15 ± 5

19 ± 4

121 ± 14

11 ± 3

50

20 ± 7

13 ± 3

11 ± 2

114 ± 12

9 ± 3

167

22 ± 7

13 ± 5

15 ± 4

112 ± 9

10 ± 3

500

23 ± 10

15 ± 3

16 ± 3

109 ± 6

12 ± 1

1667

23 ± 8

13 ± 2

14 ± 3

117 ± 5

13 ± 2

5000

22 ± 10

11 ± 1

13 ± 2

99 ± 13

14 ± 2

 

Table 2. Preincubation Method: Mean Number of Revertant Colonies in the Presence and Absence of S9 Mix

Substance

Concentrations ( µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

In the Presence of S9

DMSO

100 µL

20 ± 3

9 ± 1

40 ± 1

139 ± 14

10 ± 1

Test Material

17

17 ± 5

16 ± 4

39 ± 8

146 ± 17

12 ± 3

50

21 ± 5

12 ± 2

41 ± 5

144 ± 16

15 ± 5

167

20 ± 3

9 ± 4

33 ± 6

149 ± 10

13 ± 1

500

19 ± 7

10 ± 1

38 ± 3

164 ± 7

12 ± 3

1667

16 ± 3

10 ± 1

37 ± 6

134 ± 8

12 ± 3

5000

20 ± 3

7 ± 2

41 ± 6

125 ± 12

13 ± 1

In the Absence of S9

DMSO

100 µL

20 ± 5

9 ± 1

26 ± 2

142 ± 11

11 ± 3

Test Material

17

15 ± 3

8 ± 1

29 ± 5

125 ± 11

11 ± 6

50

18 ± 5

13 ± 3

25 ± 7

124 ± 13

12 ± 3

167

17 ± 2

8 ± 3

23 ± 3

130 ± 3

9 ± 3

500

19 ± 2

10 ± 3

27 ± 5

121 ± 9

10 ± 4

1667

18 ± 6

9 ± 4

31 ± 3

122 ± 9

8 ± 4

5000

19 ± 2

6 ± 2

20 ± 2

136 ± 5

10 ± 4

 

Table 3. Positive Control Results

Test Method

Presence/Absence of S9 mix

Compound

Strain

Concentration (µg/plate)

Colony Count (mean ± SD)

Direct Plate Method

+

2AAN

TA 1535

2

608 ± 40

TA 1537

2

705 ± 55

TA 98

0.5

235 ± 29

TA 100

0.5

1136 ± 71

WP2uvrA

20

701 ± 31

-

NaN₃

TA 1535

1

335 ± 80

9AA

TA 1537

80

2945 ± 276

2NF

TA 98

1

468 ± 30

NaN₃

TA 100

1

938 ± 61

ENNG

WP2uvrA

2

864 ± 26

Preincubation Method

+

2AAN

TA 1535

2

465 ± 32

TA 1537

2

441 ± 6

TA 98

0.5

955 ± 34

TA 100

0.5

1994 ± 23

WP2uvrA

20

673 ± 52

-

NaN₃

TA 1535

1

490 ± 26

9AA

TA 1537

80

4048 ± 546

2NF

TA 98

1

723 ± 73

NaN₃

TA 100

1

1160 ± 24

ENNG

WP2uvrA

2

902 ± 60

2-AAN = 2-Aminoanthracene

NaN₃ = Sodium azide

9AA = 9-Aminoacridine

2NF = 2-Nitrofluorene

ENNG = N-Ethyl-N-nitro-N-nitrosoguanidine

Table 4. Toxicity Test

Strain

Concentration (µg/plate)

S-9 mix

Revertant Colony Count

TA 100

DMSO

-

114

17

-

112

50

-

122

167

-

114

500

-

119

1667

-

115

5000

-

96

DMSO

+

121

17

+

121

50

+

142

167

+

122

500

+

132

1667

+

112

5000

+

102

Applicant's summary and conclusion

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

Under the conditions of the test, no mutagenic response was induced in any of the five strains tested in either the presence or absence of metabolic activation.
Executive summary:

In a GLP compliant study performed to standardised guidelines, the mutagenic potential of the test material was assessed in an Ames test. Both S. typhimurium and E. coli were exposed to the test material by both direct plate application and preincubation methods. The following strains were tested, with and without the presence of metabolic activation by S9 -mix: TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA. Positive and solvent controls were run concurrently.

Under the conditions of the test, no mutagenic response was observed in any of the five strains tested in either the presence or absence of metabolic activation up to a maximum concentration of 5000 µg per plate. The results obtained in both mutation assays were similar. There was no toxicity to the bacteria and no precipitation of the test material at any of the concentrations tested. Positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. All results were valid with the exception of the counts obtained for TA 100 with 9-aminoacridine. The mutant counts were higher than those reported in the historical data. However this was not considered to have affected the integrity of the study results.