Methomyl

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:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 59 NohSan No. 4200

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methomyl

EC Number:

240-815-0

EC Name:

Methomyl

Cas Number:

16752-77-5

Molecular formula:

C5H10N2O2S

IUPAC Name:

(E)-[1-(methylsulfanyl)ethylidene]amino N-methylcarbamate

Test material form:

solid: crystalline

Specific details on test material used for the study:

- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-394
- Purity: 98.35%

Method

Target gene:

his, trp

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA97a, and TA98

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Male SD rat liver homogenate activation system (S9 Mix)

Test concentrations with justification for top dose:

Trial I: 10, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for TA100 and WP2 uvrA (pKM101)
10, 50, 100, 500, 1000, 2500, and 5000 μg/plate for TA97a, TA98, and TA1535
Highest concentration is the guideline limit dose for this test system
Trial II: 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate for all strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance appeared completely soluble at 50 mg/mL producing a solution immediately upon mixing. Concentrations were calculated with the assumption that addition of the test substance to the solvent did not change the volume of the resulting solution.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, ICR 191 Acridine

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)
- Cell density at seeding: At least 1E+8 bacteria

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Overnight culture

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the background lawn

Evaluation criteria:

A test substance was classified as positive when: (1) the average number of revertants in any strain at any test substance concentration studied was at least two times greater than the average number of revertants in the negative control; and (2) there was a positive doseresponse relationship in that same strain.

A test substance was classified as negative when either: (1) there were no test substance concentrations with an average number of revertants which was at least two times greater than the average number of revertants in the negative control; and (2) there was no positive doseresponse relationship.

Results not meeting these criteria for positive or negative assessments were evaluated using scientific judgment.

Statistics:

Data for each tester strain were evaluated independently. For each tester strain, the average number of revertants and the standard deviation at each concentration with and without S9 activation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA100, TA1535, TA97a, and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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: Mutagenic activity of the test substance in strain TA100 in Trail 2

Without activation
Concentration of test substance (µg/plate)	Revertants			Average	SD
	Plate 1	Plate 2	Plate 3
0.0	99	104	107	103	4
10.0	86	96	113	98	14
50.0	108	118	113	113	5
100.0	110	107	92	103	10
500.0	94	92	99	95	4
1000.0	92	105	99	99	7
2500.0	118	91	104	104	14
5000.0	103	86	84	91	10
Sodium azide (2 µg/plate)	891	985	994	957	57
With activation
0.0	93	99	107	100	7
10.0	95	115	107	106	10
50.0	108	108	93	103	9
100.0	97	101	97	98	2
500.0	103	95	99	99	4
1000.0	107	111	93	104	9
2500.0	89	92	101	94	6
5000.0	98	80	91	90	9
2AA (1 µg/plate)	984	920	902	935	43

Table-2: Mutagenic activity of the test substance in strain TA1535 in Trail 2

Without activation
Concentration of test substance (µg/plate)	Revertants			Average	SD
	Plate 1	Plate 2	Plate 3
0.0	21	18	23	21	3
10.0	17	20	14	17	3
50.0	18	13	11	14	4
100.0	14	22	15	17	4
500.0	13	12	15	13	2
1000.0	13	19	19	17	3
2500.0	19	13	22	18	5
5000.0	17	16	17	17	1
Sodium azide (2 µg/plate)	673	799	793	755	71
With activation
0.0	17	18	17	17	1
10.0	19	12	14	15	4
50.0	14	15	15	15	1
100.0	18	14	10	14	4
500.0	12	17	13	14	3
1000.0	14	11	17	14	3
2500.0	19	14	16	16	3
5000.0	17	15	11	14	3
2AA (1 µg/plate)	314	332	328	325	9

Table-3: Mutagenic activity of the test substance in strain TA97a in Trail 2

Without activation
Concentration of test substance (µg/plate)	Revertants			Average	SD
	Plate 1	Plate 2	Plate 3
0.0	125	131	138	131	7
10.0	121	121	125	122	2
50.0	143	135	128	135	8
100.0	132	125	124	127	4
500.0	119	120	112	117	4
1000.0	116	133	137	129	11
2500.0	125	118	117	120	4
5000.0	118	114	126	119	6
ICR 191 (2 µg/plate)	1912	1745	1914	1857	97
With activation
0.0	140	124	137	134	9
10.0	121	127	125	124	3
50.0	127	123	126	125	2
100.0	133	131	130	131	2
500.0	133	136	143	137	5
1000.0	120	137	129	129	9
2500.0	132	135	126	131	5
5000.0	108	106	101	105	4
2AA (1 µg/plate)	1057	1050	1048	1052	5

Table-4: Mutagenic activity of the test substance in strain TA98 in Trail 2

Without activation
Concentration of test substance (µg/plate)	Revertants			Average	SD
	Plate 1	Plate 2	Plate 3
0.0	29	29	28	29	1
10.0	25	28	21	25	4
50.0	22	23	26	24	2
100.0	31	26	36	31	5
500.0	32	34	35	34	2
1000.0	27	21	26	25	3
2500.0	40	33	27	33	7
5000.0	29	28	38	32	6
2NF (25 µg/plate)	1327	1570	1439	1445	122
With activation
0.0	34	31	35	33	2
10.0	39	36	38	38	2
50.0	41	37	41	40	2
100.0	44	37	43	41	4
500.0	44	39	44	42	3
1000.0	34	36	31	34	3
2500.0	36	41	27	35	7
5000.0	34	28	37	33	5
2AA (2 µg/plate)	1838	1590	1789	1739	131

Table-5: Mutagenic activity of the test substance in strain WP2 uvrA (PKM101) in Trail 2

Without activation
Concentration of test substance (µg/plate)	Revertants			Average	SD
	Plate 1	Plate 2	Plate 3
0.0	192	189	178	186	7
10.0	204	182	190	192	11
50.0	182	188	187	186	3
100.0	210	187	190	196	13
500.0	218	205	190	204	14
1000.0	202	188	185	192	9
2500.0	203	213	205	207	5
5000.0	217	204	194	205	12
MMS (1000 µg/plate)	1985	1834	2183	2001	175
With activation
0.0	205	175	187	189	15
10.0	165	208	208	194	25
50.0	206	194	186	195	10
100.0	213	179	197	196	17
500.0	200	189	206	198	9
1000.0	192	196	195	194	2
2500.0	201	201	208	203	4
5000.0	196	171	194	187	14
2AA (25 µg/plate)	1878	1907	2141	1975	144

Applicant's summary and conclusion

Conclusions:

Negative in Salmonella typhimurium strains and in Escherichia coli strain with and without an exogenous metabolic activation system (S9)

Executive summary:

The test substance, was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98 and in Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9). The study was conducted according to OECD guidelines 471 and 472, U.S. EPA 84-2.

Concentrations of 10, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate were initially evaluated using Salmonella typhimurium strain TA100 and Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9). In the absence of any notable bacterial toxicity or test substance precipitation, concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate were subsequently tested in Salmonella typhimurium strains TA97a, TA98, and TA1535 to complete the first trial.

In a second confirmatory trial, concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate were tested in comparison to negative (solvent) controls in all strains.

Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. The test substance was negative.