3,5-dimethylpyrazole

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:

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 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:

other: EPA 712-C-98-247

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: CPMP/ICH/141/95

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: CPMP/ICH/174/95

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

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.