Methomyl

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

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:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

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:

Methomyl technical
Lot No.: DPX-X1179-512
Purity: 98.8%

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Fredrick, Maryland, U.S.A.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 9 weeks old
- Weight at study initiation: 19.3-24.3 g
- Housing: Animals were housed 1 group per plastic shoebox cage with appropriate bedding
- Diet: LLC Certified Rodent LabDiet# 5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: Quarantined for 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18-26°C
- Humidity: 30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 5, 25, 50 and 100%
Positive control: 0 and 25%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- weighed 3 times during quarantine and 4 times prior to initiation of dosing
- observed with respect to weight gain and any gross signs of disease or injury

MAIN STUDY : 25 µL of the test substance were administered topically to the dorsum of each mouse ear for 3 consecutive days (test days 0-2). One group of mice was similarly dosed with the positive control and one group of mice was similarly dosed with the positive control vehicle. Test days 3-4 were days of rest followed by intravenous injection of 20 µCi of 3H-Thymidine per mouse on test day 5. Approximately 5 hours after the injection, animals were sacrificed by carbon dioxide asphyxiation, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were incubated at 2-8°C overnight. On test day 6, the single cell suspensions were counted on a beta counter. The counts per minute (cpm) data were convened to disintegrations per minute (dpm).
A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group.

ANIMAL ASSIGNMENT AND TREATMENT
- Mice, selected based on adequate body weight gain and freedom from any ear abnormalities (e.g. torn, scratched) or clinical signs of disease or injury, distributed into study groups. Prior to study start, each mouse was assigned to a group using a randomly generated, computer-based algorithm such that individual pretest body weights did not vary more than 20% of the group mean.
- Criteria used to consider a positive response: Statistically significant increases in cell proliferation in the test concentration groups compared to the vehicle control group and/or SI's of greater than or equal to 3.0 indicated a positive response.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

- Bodyweight, bodyweight gain and Lymph node dpm data: If the Shapiro-Wilk test was not significant but Levene's test was significant, a robust version of Dunnett's test was used. If the Shapiro-Wilk test was significant, Kruskal-Wallis test was followed by Dunn's test.
Positive control and positive control vehicle data were not included in the statistical analysis of the test substance groups.
- Incidence of clinical observations: If the incidence was not significant, but a significant lack of fit occurred, then Fisher's Exact test with a Bonferroni correction was used.

Results and discussion

Positive control results:

A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.54

Test group / Remarks:

5%

Results 2

Key result

Parameter:

SI

Value:

0.35

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

0.23

Test group / Remarks:

50%

Results 4

Parameter:

SI

Value:

6.9

Test group / Remarks:

25% HCA (positive control)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : No statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at any test concentration.

DETAILS ON STIMULATION INDEX CALCULATION & EC3 CALCULATION : Stimulation indexes of less than 3.0 were observed at all test concentrations of the test substance. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., stimulation index = 3) for the test substance under the conditions of this study was not calculable.

CLINICAL OBSERVATIONS: No clinical signs of toxicity were observed in the study.

BODY WEIGHTS: A statistically significant decrease in mean body weight compared to the vehicle control group was observed in the 25% test concentration at test day 5. No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any other test concentration.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test substance did not produce a dermal sensitization response in mice.

Executive summary:

The objective of this study was to evaluate the potential of the test substance to produce a dermal sensitization response in mice using the local lymph node assay (LLNA) following OECD guideline 429 and U.S. EPA OPPTS 870.2600. Five groups of 5 female mice were dosed for 3 consecutive days with 0 (vehicle control), 5, 25, 50, or 100% test substance on both ears. N,N-dimethyIformamide was used as the diluting vehicle. One group of 5 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in 4:1 acetone:olive oil (AOO) as a positive control and one group of 5 female mice was dosed for 3 consecutive days with AOO as a positive control vehicle. On test day 5 of the assay, mice received 3H-Thymidine by tail vein injection and were sacrificed approximately 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears from the test substance groups was then evaluated and compared to the vehicle control group.

A statistically significant decrease in mean body weight compared to the vehicle control group was observed in the 25% test concentration at test day 5. No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any other test concentration. Six mice (606 and 607 in the 25% test substance group, and 1006, 1008, 1009, and 1010 in the 100% test substance group) were found dead on test day 1. Five of these mice (606, 607, 1006, 1009, and 1010) had no abnormality detected at necropsy. The remaining mouse (1008) had red lung discoloration at necropsy. One mouse (1007 in the 100% test substance group) was removed from study on test day 2 due to discontinuation of dosing that group. No clinical signs of toxicity were observed in the study. Given that the oral LD50 in rats is 32 mg/kg and that the mice in the 100% dose group received a dermal application of approximately 2500 mg/kg, which was not occluded, the observed deaths may be attributed to ingestion of the test material by the mice due to grooming.

No statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at any test concentration. Stimulation indexes of less than 3.0 were observed at all test concentrations of the test substance. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., stimulation index - 3) for the test substance under the conditions of this study was not calculable. Based on these data, the test substance is not a dermal sensitizer.

A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study with the test substance. Under the conditions of this study, the test substance did not produce a dermal sensitization response in mice.