Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Study Type  Species Findings  Guideline Reliability 
LLNA Mice Not a sensitizer    

OECD 429, OPPTS 870.2600 

 1
Buehler test Guinea Pigs Not a sensitizer  OECD 406, OPPTS 870.2600  1
Buehler test Guinea Pigs Not a sensitizer  OECD 406, EPA 81-6  1

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF 12-Nouan-8147 (2000)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Specific details on test material used for the study:
Methomyl Technical
Lot No.: DPX-X1179-599
Purity: 99.6%
Species:
guinea pig
Strain:
other: Hartley albino
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elm Hill Breeding Laboratories, Chelmsford, Massachusetts
- Age at study initiation: Young adult
- Weight at study initiation: 361-442 g
- Housing: Animals were group housed in suspended stainless steel with mesh floors or plastic perforated bottom caging. Litter paper was placed beneath the cage and was changed at least three times per week.
- Diet: Pelleted Purina Guinea Pig Chow #5025
- Water: Filtered tap water, ad libitum
- Acclimation period: 7 or 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-22ºC
- Humidity: 39-64%
- Photoperiod (hrs dark / hrs light): 12/12
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
0.4 g of 75% w/w mixture of the ground test substance in mineral oil
Day(s)/duration:
6 hours, once each week for a three week induction period
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
0.4 g of 75% w/w mixture of the ground test substance in mineral oil
Day(s)/duration:
6 hours exposure (27 days after the first induction dose)
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Preliminary Irritation Testing: 4 per group
Test group: 20 per group
Congrol group: 10 per group
Details on study design:
Preliminary Irritation Testing: A group of animals was used to determine the highest non-irritating concentration (HNIC) of the test substance prior to the challenge dose. The ground test substance was applied mixed with mineral oil to yield w/w concentrations of 75%, 56%, 38% and 19%. Each concentration was applied (0.4 g or mL) to a test site using an occlusive 25 mm Hill Top Chamber and applied to the appropriate test site. The sites were wrapped with non-allergenic Durapore adhesive tape. After six hours of exposure, the chambers were removed and the test sites were gently cleansed of any residual test substance. Approximately 24 hours after application, each site was evaluated for local reactions (erythema). From these results, the HNIC (the highest concentration that produced responses in 4 guinea pigs no more severe than two scores of 0.5 and two scores of zero) was established and used for challenge. The HNIC selected for the challenge phase was a 75% w/w mixture in mineral oil.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Once each week for three weeks
- Exposure period: 6 hours
- Test groups: One group with 20 animals
- Site: Left side of midline
- Concentrations: 75% w/w mixture of the ground test substance in mineral oil

B. CHALLENGE EXPOSURE
- No. of exposures: One
- Exposure period: 6 hours
- Test groups: One group with 20 animals
- Control group: One group with 10 animals
- Site: Right front flank
- Concentrations: 75% w/w mixture of the ground test substance in mineral oil
- Evaluation (hr after challenge): Approximately 24 and 48 hours
Challenge controls:
Mineral oil
Positive control substance(s):
no
Remarks:
Procedures used in this study were validated historically using alpha-Hexylcinnamaldehyde, technical grade (HCA) as a positive control substance.
Positive control results:
Historical Positive Control Animals (100% HCA): All positive control animals exhibited signs of a sensitization response (faint to moderate erythema [1-2]) 24 hours after challenge. Similar indications persisted at four of these sites through 48 hours. Very faint erythema (0.5) was noted for most other sites following challenge
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75% w/w
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75% w/w
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the findings the test substance is considered not to be a contact sensitizer.
Executive summary:

A dermal sensitization test was conducted with guinea pigs to determine the potential for the test substance to produce sensitization after repeated topical applications following the guidelines OECD 406 and EPA OPPTS 870.2600.

Preliminary irritation testing was performed on four animals to determine appropriate concentrations of the test substance that could be used for both the induction and challenge phases.

A 75% w/w mixture of the ground test substance in mineral oil was topically applied for six hours to 20 healthy test guinea pigs, once each week for a three week induction period. Twenty-seven days after the first induction dose, a challenge dose of the test substance at its highest non-irritating concentration (HNIC, determined in the preliminary irritation screen to be a 75% w/w mixture in mineral oil) was applied to a naive site on each of the test guinea pigs. A naive control group (ten animals) was maintained under the same environmental conditions and treated with the test substance at challenge only. Approximately 24 and 48 hours after each induction and challenge dose, the test and naive control animals were scored for erythema.

Very faint erythema (0.5) was noted for three of twenty test sites 24 hours after challenge and very faint erythema (0.5) was noted for one of ten naive control sites 24 hours after challenge in the naive control animals. Irritation cleared from the affected sites by 48 hours in the test animals.

Based on the results of this study, the test substance is considered not to be a contact sensitizer. The positive response observed in the historical positive control validation study with alpha-hexylcinnamaldehyde technical (HCA) validates the test system used in this study.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Specific details on test material used for the study:
Methomyl Technical
Lot No.: DPX-X1179-394
Purity: 98.35%
Species:
guinea pig
Strain:
other: Duncan Hartley albino
Sex:
male/female
Route:
epicutaneous, occlusive
Vehicle:
other: distilled water
Concentration / amount:
300 mg of neat test substance moistned in 0.3 ml of distilled water
Day(s)/duration:
6 hours, once each week for a three week induction period
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: distilled water
Concentration / amount:
0.3 ml of neat test substance moistned in 0.3 ml of distilled water
Day(s)/duration:
6 hours exposure (two weeks after the last induction dose)
No. of animals per dose:
Test group: 10 per sex per group
Congrol group: 5 per sex per group
Challenge controls:
Acetone
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene (DNCB)
Positive control results:
Slight to severe erythema was observed in the positive control animals during the challenge phase.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Neat test substance
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Neat test substance
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the test substance, did not produce delayed contact hypersensitivity in guinea pigs.
Executive summary:

The dermal sensitization test was conducted with guinea pigs following the guidelines OECD 406 and EPA OPP 81-6. The test substance dosed as received (moistened with distilled water) was tested on the clipped, intact skin of 10 male and 10 female guinea pigs 1-chloro-2,4-dinitrobenzene (DNCB) was used to demonstrate the ability of the test system to detect sensitizers (positive control group). The vehicle (80% ethanol) was administered to 5 male and 5 female guinea pigs (negative control).

During the induction phase, no erythema was observed in the test substance treated animals. Slightly patchy to severe erythema was observed in the positive control animals during the induction phase. No erythema was observed during the three inductions in the negative control animals. During the challenge phase, no signs of erythema were observed in the animals treated with the test substance at 24 or 48 hours. Slight to severe erythema was observed in the positive control animals. No signs of erythema were observed in the negative control animals at the vehicle (acetone) or test substance treated sites.

Under the conditions of this study, the test substance, did not produce delayed contact hypersensitivity in guinea pigs.

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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
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)
Specific details on test material used for the study:
Methomyl technical
Lot No.: DPX-X1179-512
Purity: 98.8%
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
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.
Positive control results:
A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice.
Key result
Parameter:
SI
Value:
0.54
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
0.35
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
0.23
Test group / Remarks:
50%
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.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The dermal sensitization potential of the test substance was evaluated in mice via the LLNA assay and in guinea pigs by the Buehler test. In the mouse study, all animals died at the top dose (2500 mg/kg) and was attributed to ingestion of the test material via grooming. 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. In one of the guinea pig studies, very faint erythema (0.5) was noted for three of twenty test sites 24 hours after challenge and very faint erythema (0.5) was noted for one of ten naive control sites 24 hours after challenge in the naive control animals. Irritation cleared from the affected sites by 48 hours in the test animals. No irritation or sensitization was observed in the second guinea pig study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

There was no evidence of dermal sensitisation in guinea pigs or mice. The substance does not need to be classified for skin sensitisation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.