Pyrasulfotole

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:

28 Aug 2003 - 24 Feb 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997 (corrected 26 Jun 2020)

Deviations:

yes

Remarks:

No details of which mutagen(s) (aside from 2-aminoanthracene) were used to demonstrate efficacy of the S9-mix.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-[2-methanesulfonyl-4-(trifluoromethyl)benzoyl]-1,3-dimethyl-1H-pyrazol-5-ol

EC Number:

609-256-3

Cas Number:

365400-11-9

Molecular formula:

C14H13F3N2O4S

IUPAC Name:

4-[2-methanesulfonyl-4-(trifluoromethyl)benzoyl]-1,3-dimethyl-1H-pyrazol-5-ol

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

16, 50, 158, 500, 1581, 5000 µg/plate with and without metabolic activation
The test substance showed bacteriotoxic effects starting at 500 µg/plate using the plate incorporation method while no bacteriotoxic effects were observed up to 5000 µg/tube using the preincubation method. Nevertheless all doses could be used for assessment purposes and therefore 5000 µg/plate represents the top dose (according OECD 471).

Vehicle / solvent:

- Vehicle/solvent used: DMSO (0.1 mL/plate)
- Justification for choice of solvent/vehicle: The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments. In DMSO AE 0317309 formed colorless to lightbrown solutions.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: -S9: nitrofurantoin (NF; 0.2 µg/plate, TA 100), 4-nitro-1,2-phenylene diamine (4-NPDA; 10 µg/plate, TA 1537; 0.5 µg/plate, TA 98) +S9: 2-aminoanthracene (2-AA; 3 µg/plate, TA 1535, TA 100, TA 1537, TA 98, TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: other: gross appraisal of the background growth, marked and dose-dependent reduction in the mutant count, titer

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met: A reproducible and dose-related increase in mutant counts of at least one strain is observed. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation: 500, 1581 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation: 500, 1581 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation: 500, 1581 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation: 500, 1581 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation: 500, 1581 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation up to the highest test substance concentration was observed in the medium.

HISTORICAL CONTROL DATA
All results were within the range of historical control data (see attachment 1 "Attached background material" for historical control data).

Any other information on results incl. tables

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

With or without S9-Mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)
		TA 1535	TA 100	TA 1537	TA 98	TA 102
-	0 (DMSO)	17 ± 2	99 ± 15	7 ± 1	23 ± 3	134 ± 28
-	16	17 ± 2	109 ± 8	6 ± 2	17 ± 3	132 ± 20
-	50	17 ± 3	109 ± 12	6 ± 1	19 ± 6	144 ± 15
-	158	12 ± 4	122 ± 34	5 ± 1	22 ± 8	163 ± 3
-	500	14 ± 5	103 ± 11	6 ± 2	19 ± 5	112 ± 13
-	1581	14 ± 1	115 ± 2	9 ± 2	24 ± 4	122 ± 8
-	5000	13 ± 2	113 ± 10	9 ± 2	17 ± 2	116 ± 14
Positive controls, - S9	Name	Na-azide	NF	4-NPDA	4-NPDA	MMC
	Concentrations [µg/plate]	10	0.2	10	0.5	0.2
	Mean No. of revertant colonies/ plate (average of 3 ± SD)	356 ± 22	349 ± 46	109 ± 25	161 ± 12	591 ± 11
+	0 (DMSO)	13 ± 3	110 ± 9	11 ± 2	30 ± 2	158 ± 8
+	16	12 ± 6	117 ± 3	8 ± 1	30 ± 1	151 ± 20
+	50	8 ± 1	114 ± 10	8 ± 4	24 ± 3	161 ± 12
+	158	9 ± 2	133 ± 39	9 ± 2	33 ± 10	175 ± 14
+	500	9 ± 1	107 ± 2	9 ± 2	34 ± 3	160 ± 9
+	1581	8 ± 2	95 ± 13	6 ± 1	30 ± 3	156 ± 14
+	5000	9 ± 2	94 ± 9	5 ± 1	18 ± 6	150 ± 23
Positive controls, + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations [µg/plate]	3	3	3	3	3
	Mean No. of revertant colonies/ plate (average of 3 ± SD)	145 ± 15	1642 ± 27	261 ± 14	1389 ± 30	542 ± 44

Na-azide: sodium azide

NF: nitrofurantoin

4-NPDA: 4-nitro-1,2-phenylene diamine

MMC: mitomycin C

2-AA: 2-aminoanthracene

 Table 2. Test results of Experiment 2 (preincubation)

With or without S9-Mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)
		TA 1535	TA 100	TA 1537	TA 98	TA 102
-	0 (DMSO)	17 ± 1	138 ± 8	10 ± 1	18 ± 3	183 ± 9
-	16	15 ± 6	139 ± 10	7 ± 2	13 ± 5	189 ± 3
-	50	20 ± 2	135 ± 6	7 ± 2	15 ± 5	201 ± 17
-	158	20 ± 5	135 ± 16	9 ± 2	20 ± 5	187 ± 29
-	500	14 ± 2	134 ± 12	7 ± 2	15 ± 4	198 ± 13
-	1581	20 ± 5	131 ± 1	7 ± 3	14 ± 2	187 ± 14
-	5000	16 ± 3	141 ± 25	6 ± 1	17 ± 3	200 ± 18
Positive controls, - S9	Name	Na-azide	NF	4-NPDA	4-NPDA	Cumene
	Concentrations [µg/plate]	10	0.2	10	0.5	10
	Mean No. of revertant colonies/ plate (average of 3 ± SD)	392 ± 17	440 ± 49	100 ± 19	142 ± 12	509 ± 23
+	0 (DMSO)	15 ± 4	181 ± 19	12 ± 2	30 ± 6	245 ± 10
+	16	16 ± 4	173 ± 10	10 ± 2	29 ± 8	212 ± 20
+	50	16 ± 3	157 ± 15	10 ± 5	23 ± 6	237 ± 16
+	158	15 ± 5	185 ± 18	9 ± 2	27 ± 7	249 ± 37
+	500	17 ± 2	157 ± 12	8 ± 1	33 ± 4	237 ± 3
+	1581	11 ± 2	156 ± 14	9 ± 2	23 ± 5	229 ± 3
+	5000	11 ± 2	155 ± 4	10 ± 3	32 ± 5	227 ± 11
Positive controls, + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations [µg/plate]	3	3	3	3	3
	Mean No. of revertant colonies/ plate (average of 3 ± SD)	159 ± 8	1561 ± 16	255 ± 29	1331 ± 59	507 ± 39

Na-azide: sodium azide

NF: nitrofurantoin

4-NPDA: 4-nitro-1,2-phenylene diamine

Cumene: cumene hydroperoxide

2-AA: 2-aminoanthracene

Table 3: Test substance stability in DMSO

Nominal value in mg/mL	Content as a % of nominal value after storage time in hours
	0	24
0.01	110	110
250	96	97

According to these result, the test substance was stable in the vehicle at room temperature at concentrations ranging from 0.01 mg/mL to 250 mg/mL for at least twenty-four hours, a time interval, which covers the time range from preparation of the formulation to last treatment.

Applicant's summary and conclusion

Conclusions:

The study was performed according to OECD guideline 471 and compliant with GLP. Under the conditions of the assay, the test item was not mutagenic in S. typhimuirum strains TA 98, TA 100, TA 1535, TA 1537 and in TA 102 with and without metabolic activation.