picoxystrobin (ISO); methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

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:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

methyl (E)-3-methoxy-2-{2-[6-(trifluoromethyl)pyridin-2-yloxymethyl]phenyl}acrylate

EC Number:

601-478-9

Cas Number:

117428-22-5

Molecular formula:

C18H16F3NO4

IUPAC Name:

methyl (E)-3-methoxy-2-{2-[6-(trifluoromethyl)pyridin-2-yloxymethyl]phenyl}acrylate

Test material form:

solid

Details on test material:

Purity: 93.3% w/w

Method

Target gene:

histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2

Remarks:

pKM101

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from phenobarbital/ß-naphthaflavone-induced SD rats

Test concentrations with justification for top dose:

100, 200, 500, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

Dimethylsulphoxide

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulphoxide

Positive controls:

yes

Positive control substance:

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

mitomycin C

other: Acridine mutagen ICR191 (TA1537/without activation), 2-Aminoanthracene (all strains/with activation), Daunomycin HCl (TA98/without activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Top agar (plate incorporation)
- Cell density at seeding: 0.1 mL aliquots of each bacterial strain

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 3 days
- All plates were counted by an automatic colony counter with the discrimination adjusted appropriately to permit the optimal counting of mutant colonies

Evaluation criteria:

A positive response in a valid individual experiment is achieved when one or both of the following criteria are met: A statistically significant dose-related increase in the mean number of revertant colonies is obtained; a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) which is statistically significant, is observed at one or more concentrations.

A negative result in a valid individual experiment is achieved when: There is no statistically significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.

Statistics:

An assessment of statistical significance was carried out using a one-tailed Student's t-test. The corresponding probability for each dose level was derived by computer using the appropriate degrees of freedom. Values of p <0.01 are treated as significant, with values of 0.01≤ p <0.05 being indicative of a possible effect.

Results and discussion

Test results

Key result

Species / strain:

other: all species/strains tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table-1: Test Data for Experimental Phase – 1

Strain	Metabolic activation	Dose levels (µg/plate)	Mean	Standard deviation	Ratio: Test/control	No Revertants/plate
						Plate 1	Plate 2	Plate 3
TA1535	+S9	5000	15.3	1.2	1.2*	16	14	16
		2500	10.0	7.8	0.8	6	19	5
		1000	13.7	6.7	1.1	18	17	6
		500	14.0	7.2	1.1	6	20	16
		200	10.0	2.6	0.8	13	9	8
		100	13.7	3.8	1.1	11	12	18
TA1535	-S9	5000	6.3	1.5	0.8	8	6	5
		2500	12.0	5.3	1.4	10	10	8
		1000	9.0	7.9	1.1	6	18	3
		500	6.0	4.0	0.7	6	2	10
		200	11.7	1.5	1.4*	10	12	13
		100	8.3	0.6	1.0	8	8	9
TA1537	+S9	5000	3.3	1.5	1.1	5	2	3
		2500	2.3	0.6	0.8	2	2	3
		1000	3.0	1.0	1.0	4	2	3
		500	5.0	1.0	1.7**	6	5	4
		200	5.0	2.6	1.7	8	3	4
		100	4.3	1.5	1.4	4	6	3
TA1537	-S9	5000	2.3	0.6	0.6	3	2	2
		2500	3.0	1.7	0.8	5	2	2
		1000	3.3	1.2	0.8	2	4	4
		500	3.7	1.5	0.9	4	5	2
		200	3.7	2.1	0.9	2	3	6
		100	3.3	0.6	0.8	3	3	4
TA98	+S9	5000	11.7	2.1	0.6	14	11	10
		2500	20.7	3.1	1.0	20	18	24
		1000	17.3	0.6	0.8	17	18	17
		500	18.3	2.5	0.9	21	16	18
		200	18.3	2.1	0.9	20	16	19
		100	20.3	4.5	1.0	20	25	16
TA98	-S9	5000	17.0	1.0	0.9	18	17	16
		2500	15.0	4.6	0.8	19	10	16
		1000	14.0	3.0	0.7	11	14	17
		500	17.0	1.0	0.9	18	16	17
		200	18.7	4.6	1.0	16	16	24
		100	20.7	3.8	1.1	18	25	19
TA100	+S9	5000	104.0	12.0	1.2*	104	116	92
		2500	77.0	5.3	0.9	73	75	83
		1000	85.0	5.3	1.0	89	79	87
		500	99.3	7.6	1.1*	91	106	101
		200	96.5	4.9	1.1	93	100	C
		100	90.7	3.5	1.0	94	91	87
TA100	-S9	5000	80.7	1.5	1.1	82	79	81
		2500	81.0	2.0	1.1	79	83	81
		1000	85.3	8.4	1.1	95	80	81
		500	81.7	8.3	1.1	75	91	79
		200	89.3	9.3	1.2*	97	79	92
		100	83.7	6.5	1.1	90	84	77
WP2P	+S9	5000	45.7	9.5	35	49	49	53
		2500	34.7	0.6	35	35	35	34
		1000	48.0	5.3	54	46	46	44
		500	47.0	3.5	49	49	49	43
		200	46.3	3.8	42	49	49	48
		100	45.0	4.4	43	42	42	50
WP2P	-S9	5000	37.3	3.2	35	36	36	41
		2500	24.3	4.0	28	20	20	25
		1000	33.7	6.5	34	40	40	27
		500	33.3	7.1	41	32	32	27
		200	34.7	4.9	38	37	37	29
		100	32.3	2.1	34	30	30	33
WP2P uvrA	+S9	5000	149.7	22.3	164	151	161	124
		2500	109.7	8.4	100	115	115	114
		1000	147.3	6.4	140	150	150	152
		500	136.3	17.2	124	156	156	129
		200	150.3	12.0	151	138	138	162
		100	143.7	2.3	145	141	141	145
WP2P uvrA	-S9	5000	93.7	12.4	86	108	108	87
		2500	76.0	17.3	57	91	91	80
		1000	104.3	3.5	108	104	104	101
		500	103.3	9.3	111	93	93	106
		200	108.7	23.3	119	125	125	82
		100	103.3	4.0	104	99	99	107

*: 0.01≤ p < 0.05, ** p <0.01 [One-sided t-Test assumes Test > Control]

C: Contaminated plate

Table 2: Test Data for Experimental Phase 2 (+S9)

Strain	Dose levels (µg/plate)	Mean	Standard deviation	Ratio: Test/control	No Revertants/plate
					Plate 1	Plate 2	Plate 3
TA1535	5000	11.0	2.6	0.9	10	14	9
	2500	18.0	1.0	1.4*	19	18	17
	1000	14.0	4.0	1.1	14	10	18
	500	13.7	2.5	1.1	14	11	16
	200	8.7	3.5	0.7	9	5	12
	100	18.3	1.2	1.5**	19	19	17
TA1537	5000	3.0	1.7	1.0	5	2	2
	2500	3.3	1.2	1.1	4	2	4
	1000	3.7	1.2	1.2	3	3	5
	500	3.7	1.5	1.2	4	2	5
	200	4.3	0.6	1.4*	4	5	4
	100	2.3	1.5	0.8	4	2	1
TA98	5000	20.3	4.5	0.8	25	20	16
	2500	20.7	2.3	0.8	18	22	22
	1000	18.3	3.2	0.7	16	22	17
	500	21.7	4.5	0.9	22	17	26
	200	19.0	2.6	0.8	22	18	17
	100	22.0	4.6	0.9	27	21	18
TA100	5000	76.3	16.5	0.9	60	93	76
	2500	82.7	4.0	1.0	82	87	79
	1000	80.7	12.0	0.9	80	69	93
	500	73.3	5.1	0.8	69	72	79
	200	85.7	6.0	1.0	85	80	92
	100	84.0	12.0	1.0	96	84	72
WP2P	5000	50.7	11.7	1.0	42	64	46
	2500	49.3	3.5	0.9	49	53	46
	1000	48.0	10.4	0.9	60	41	43
	500	53.0	4.0	1.0	57	49	53
	200	56.0	5.2	1.1	59	59	50
	100	50.0	4.4	0.9	53	45	52
WP2P uvrA	5000	155.7	8.5	1.1	162	159	146
	2500	156.0	4.4	1.1	161	154	153
	1000	150.0	4.0	1.0	146	150	154
	500	157.3	2.1	1.1*	155	159	158
	200	160.0	13.7	1.1	163	172	145
	100	147.3	14.0	1.0	148	161	133

*: 0.01≤ p < 0.05, ** p <0.01 [One-sided t-Test assumes Test > Control]

Table 3: Test Data for Experimental Phase 2 (-S9)

Strain	Dose levels (µg/plate)	Mean	Standard deviation	Ratio: Test/control	No Revertants/plate
					Plate 1	Plate 2	Plate 3
TA1535	5000	14.0	3.5	1.3	12	12	18
	2500	11.7	0.6	1.1	11	12	12
	1000	13.0	3.0	1.3	10	16	13
	500	9.0	1.7	0.9	11	8	8
	200	9.3	1.5	0.9	11	8	9
	100	9.0	3.6	0.9	13	8	6
TA1537	5000	2.3	0.6	1.0	3	2	2
	2500	2.0	1.0	0.9	3	1	2
	1000	2.7	2.9	1.2	6	1	1
	500	2.7	1.2	1.2	4	2	2
	200	2.3	1.2	1.0	3	1	3
	100	2.3	0.6	1.0	2	2	3
TA98	5000	14.7	3.2	0.6	17	11	16
	2500	18.7	2.5	0.7	16	21	19
	1000	24.3	6.0	0.9	30	18	25
	500	23.3	4.9	0.9	21	29	20
	200	20.7	6.4	0.8	28	17	17
	100	24.7	3.5	0.9	25	28	21
TA100	5000	70.3	5.5	0.9	74	73	64
	2500	72.3	3.1	0.9	73	75	69
	1000	66.3	5.5	0.9	72	61	66
	500	65.3	3.8	0.9	61	68	67
	200	66.3	3.8	0.9	68	69	62
	100	74.3	8.4	1.0	69	84	70
WP2P	5000	34.7	6.0	1.2	34	29	41
	2500	27.3	0.6	1.0	27	28	27
	1000	27.7	3.2	1.0	30	29	24
	500	30.0	3.5	1.1	34	28	28
	200	28.0	7.2	1.0	36	26	22
	100	28.3	1.2	1.0	29	27	29
WP2P uvrA	5000	133.7	16.3	1.1	148	116	137
	2500	111.3	4.5	0.9	107	111	116
	1000	121.7	3.1	1.0	119	121	125
	500	108.3	4.5	0.9	108	104	113
	200	127.3	9.1	1.0	117	134	131
	100	127.7	15.0	1.0	140	132	111

Applicant's summary and conclusion

Conclusions:

The test substance was negative (with and without activation) in all strains tested

Executive summary:

The test substance was evaluated in a bacterial mutagenicity assay over a range of concentrations using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two strains of Escherichia coli (WP2P and WP2P uvrA) in the presence and absence of a rat liver - derived metabolic activation system (S9-mix), following protocols complying with OECD Guideline Numbers 471 and 472 and with the United Kingdom Department of Health and Guidelines (DOH).

In two separate experiments, the test substance did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any of the tester strains used, either in the presence or absence of S9-mix.

The sensitivity of the test system, and the metabolic activity of the S9-mix, were clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e., non-mutagenic in S. typhimurium and E. coli strains in both the presence and absence of S9 -mix.