Methomyl

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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:

1995

Report date:

1995

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:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 4

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:

mammalian erythrocyte micronucleus test

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%

Test animals

Species:

mouse

Strain:

other: Crl:CD®-1(ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, North Carolina
- Age at study initiation: 62 days
- Weight at study initiation: 29.1-36.0 g (males); 21.5-27.3 g (females)
- Assigned to test groups randomly: Yes
- Housing: Standard wire mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 50 ± 10
- Photoperiod: 12-hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Sterile water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of the test substance in the vehicle were prepared at concentrations of 0.3, 0.6, and 1.2 mg/mL. Uniformity was maintained during dosing by constant stirring. Single acute doses of the appropriate solutions were administered by oral intubation at 10 mL/kg yielding treatments of 3, 6, and 12 mg/kg bw.
The negative control (vehicle) was administered by gavage in a volume of 10 mL/kg.

Duration of treatment / exposure:

Single oral dose

Frequency of treatment:

Single oral dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

6 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

12 mg/kg bw (total dose)

No. of animals per sex per dose:

5 (solvent control; 3, 6 mg/kg; positive control) and 6 (12 mg/kg)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CP)
- Route of administration: Oral intubation
- Doses / concentrations: 40 mg/kg

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results from the range finding study, a dose of 12 mg/kg was selected as the maximum dose for both male and female mice

SAMPLING TIMES: 24, 48, and 72 hours

DETAILS OF SLIDE PREPARATION: Immediately after sacrifice, marrow from both femurs of each animal was aspirated and flushed into approximately 2 mL prewarmed (37°C) fetal bovine serum. The marrow was collected by centrifugation. Most of the supernatant was removed, and the cells were resuspended in the remaining 1-2 drops of serum. A Miniprep® automatic blood smearing instrument was used to prepare marrow smears. At least 3 slides per animal were prepared and fixed in absolute methanol for 8 mins. Slides were stained for 2.5 mins in 0.0125 mg/mL acridine orange in phosphate buffer (pH 7.4). Prior to scoring, a coverslip was floated on each slide using phosphate buffer.

METHOD OF ANALYSIS: Representative slides from each animal were examined blindly using incident light fluorescence microscopy. Only cells with good morphology and staining were scored. Colour was used to distinguish PCEs (reddish) from NCEs (dark green). PCEs (2000 per animal) were scored for the presence of micronuclei (round, bright yellow-green fluorescing bodies). Cellular inclusions that were irregularly shaped or stained, or out of the focal plane of the cell were considered artifacts. The unit of scoring was the micronucleated cell; PCEs with more than one micronucleus were scored as a single micronucleated PCE (MNPCE). Micronucleated NCEs seen in the optic fields scored to obtain 2000 PCEs were also counted. Additionally, the number of PCEs among 1000 erythrocytes was recorded for each animal.

Statistics:

Data for the proportion of MNPCEs among 2000 PCEs and the proportion of PCEs among 1000 erythrocytes were transformed prior to analysis using the arcsin square root function. Transformed data for PCE or MNPCE frequencies were analyzed separately for normality of distribution using the Shapiro-Wilkes test. If results indicated that the transformed values for PCE or MNPCE frequencies were normally distributed in both sexes, parametric methods (viz., Analysis of Variance and Dunnett test) were used. If there was nonnormality in either sex, nonparametric methods (viz., Krushkal-Wallis test and Mann-Whitney U tests) were used for that variable using nontransformed proportions. Positive indicator data were not included in evaluating normality of distribution. Weight gain data were assumed to be normally distributed and were analyzed by ANOVA. Data from each sex and sacrifice time were analyzed separately, and individual comparisons to the control were made using each animal as the experimental unit. All analyses were conducted at a significance level of 5%. Positive indicator data were analyzed separately.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

1 female mouse at 12 mg/kg was found dead ~24 h after dosing. Hyperactivity was displayed by 1 male at same dose. Lethargy was seen at 6 mg/kg in 3 males and at 12 mg/kg in 1 male and 1 female mice. One of these mice had half shut eyes 24 h post-dosing.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY FOR DOSE SELECTION
- Dose range: 5 to 40 mg/kg
- Clinical signs of toxicity in test animals: Deaths occurred at 15 mg/kg (1/4 males, 0/4 females), 20 mg/kg (3/5 males, 2/6 females), 25 mg/kg (2/2 females), 30 mg/kg (4/5 males, 7/7 females), and 40 mg/kg (3/3 males, 3/3 females). Animals dosed at 20, 25, 30, and 40 mg/kg exhibited tremors, convulsions, and/or gasping within 30 minutes post-exposure. These symptoms lasted up to approximately seven minutes until the animals either died or recovered. Surviving animals then became lethargic and quiet. By 3 hours post-dosing, no signs of toxicity were observed. At 15 mg/kg, the surviving mice were lethargic and had shallow respiration immediately following dosing which subsided within the following hour. No deaths or clinical signs were observed at 5 or 10 mg/kg.

Any other information on results incl. tables

Table-1: PCE Frequency

Test substance (mg/kg)	Sampling time (hrs)	Sex	N	Mean %PCE (95% conf. limits) (determined on transformed values)	Mean PCE/NCE ratio (SEM)
0	24	M	5	56.3 (44.0, 68.2)	1.38 (0.24)
0	24	F	5	56.4 (48.1, 64.6)	1.35 (0.20)
3	24	M	5	61.2 (55.9, 66.3)	1.60 (0.12)
3	24	F	5	59.4 (47.3, 70.8)	1.58 (0.33)
6	24	M	5	57.4 (43.2, 70.9)	1.51 (0.41)
6	24	F	5	52.1 (48.1, 56.2)	1.10 (0.06)
12	24	M	6	45.0 (38.3, 51.7)	0.84 (0.08)
12	24	F	5^a	53.1 (48.7, 57.6)	1.14 (0.08)
0	48	M	5	47.1 (41.6, 52.6)	0.90 (0.07)
0	48	F	5	53.0 (45.8, 60.2)	1.16 (0.14)
3	48	M	5	47.3 (39.3, 55.3)	0.92 (0.11)
3	48	F	5	50.9 (39.2, 62.6)	1.10 (0.19)
6	48	M	5	51.4 (41.6, 61.1)	1.10 (0.15)
6	48	F	5	53.7 (47.4, 59.9)	1.18 (0.11)
12	48	M	6	49.5 (40.2, 58.9)	1.03 (0.15)
12	48	F	5^b	53.2 (49.9, 56.4)	1.14 (0.05)
0	72	M	5	52.8 (43.8, 61.6)	1.16 (0.17)
0	72	F	4^b	51.2 (43.6, 58.9)	1.07 (0.09)
3	72	M	5	43.9 (35.7, 52.2)	0.80 (0.10)
3	72	F	5	54.2 (45.7, 62.6)	1.22 (0.15)
6	72	M	5	45.4 (30.6, 60.6)	0.90 (0.18)
6	72	F	5	55.1 (45.4, 64.7)	1.28 (0.18)
12	72	M	6	43.5 (40.0, 47.0)	0.77 (0.04)
12	72	F	6	56.4 (52.2, 60.6)	1.31 (0.09)
CP, 40	24	M	5	51.5 (48.6, 54.5)	1.07 (0.05)
CP, 40	24	F	5	51.5 (42.9, 60.0)	1.10 (0.15)

a Mouse died prior to scheduled sacrifice time

b Mouse was misdosed and died within 24 hours of dosing

Table-2: MNPCE Frequency

Test substance (mg/kg)	Sampling time (hrs)	Sex	N	%MNPCEs
				Mean (SEM)	Median (IQR)
0	24	M	5	0.22 (0.06)	0.30 (0.25)
0	24	F	5	0.14 (0.03)	0.15 (0.12)
3	24	M	5	0.32 (0.07)	0.30 (0.25)
3	24	F	5	0.16 (0.08)	0.15 (0.32)
6	24	M	5	0.30 (0.05)	0.30 (0.20)
6	24	F	5	0.15 (0.02)	0.15 (0.10)
12	24	M	6	0.20 (0.04)	0.22 (0.14)
12	24	F	5^a	0.21 (0.06)	0.20 (0.18)
0	48	M	5	0.14 (0.04)	0.15 (0.18)
0	48	F	5	0.17 (0.03)	0.15 (0.10)
3	48	M	5	0.18 (0.05)	0.15 (0.18)
3	48	F	5	0.15 (0.06)	0.15 (0.25)
6	48	M	5	0.18 (0.05)	0.20 (0.20)
6	48	F	5	0.14 (0.05)	0.15 (0.22)
12	48	M	6	0.17 (0.04)	0.18 (0.19)
12	48	F	5^b	0.18 (0.06)	0.10 (0.25)
0	72	M	5	0.16 (0.06)	0.15 (0.22)
0	72	F	4^b	0.14 (0.04)	0.12 (0.16)
3	72	M	5	0.11 (0.03)	0.10 (0.12)
3	72	F	5	0.15 (0.03)	0.15 (0.10)
6	72	M	5	0.10 (0.03)	0.10 (0.10)
6	72	F	5	0.16 (0.04)	0.15 (0.18)
12	72	M	6	0.13 (0.02)	0.12 (0.11)
12	72	F	6	0.19 (0.04)	0.18 (0.15)
CP, 40	24	M	5	1.97 (0.52)	1.85 (2.05)*
CP, 40	24	F	5	2.07 (0.25)	1.90 (0.92)*

a Mouse died prior to scheduled sacrifice time

b Mouse was misdosed and died within 24 hours of dosing

* p ≤0.05

Applicant's summary and conclusion

Conclusions:

Negative in mouse bone marrow micronucleus assay

Executive summary:

The test substance was evaluated for its ability to induce micronuclei in bone marrow polychromatic erythrocytes (PCEs) of mice. The study was conducted following OECD guideline 474 and U.S. EPA 84-2.

A single acute dose of 0, 3, 6, or 12 mg/kg was administered by oral intubation to groups of male and female mice. In the vehicle control and all test substance treated groups, bone marrow smears were prepared approximately 24, 48, and 72 hours after dosing. 2000 PCEs per animal were scored for micronuclei.

No statistically significant increases in the frequency of micronucleated PCEs were observed in test substance treated mice at any dose level or sampling time. In addition, no statistically significant depressions in the proportion of PCEs among 1000 erythrocytes were observed. In this assay, the test substance was negative.