α-n-butyl-α-(4-chlorophenyl)-1H-1,2,4-triazole-1-propanenitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-August-2005 to 09-November-2005

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:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

α-n-butyl-α-(4-chlorophenyl)-1H-1,2,4-triazole-1-propanenitrile

EC Number:

410-400-0

EC Name:

α-n-butyl-α-(4-chlorophenyl)-1H-1,2,4-triazole-1-propanenitrile

Cas Number:

88671-89-0

Molecular formula:

C15H17ClN4

IUPAC Name:

2-(4-chlorophenyl)-2-[(1H-1,2,4-triazol-1-yl)methyl]hexanenitrile

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Remarks:

BALB/cAnNCrl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Kingston, New York)
- Age at study initiation: 8-12 weeks
- Housing: Animals were housed one per cage in stainless steel cages. Cages had wire-mesh floors that were suspended above absorbent paper.
- Diet: Animals were provided LabDiet Certified Rodent Diet in pelleted form ad libitum.
- Water: Municipal water was provided ad libitum.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 40-70%
- Air changes (per hr): Approximately 12-15 times/hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/dark

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

DMSO

Concentration:

5%, 20%, or 80% myclobutanil

No. of animals per dose:

6 females

Details on study design:

PRE-SCREEN TESTS:
- Irritation: Prior to the LLNA study, several concentrations of the test material were evaluated for irritation potential as measured by erythema of the ears. Mice (one female/group) received one application of myclobutanil (1%, 5%, 10%, 20%, 40%, or 80%) on the dorsal surface of each ear (25 ml) on two consecutive days.
- Erythema scores: No visual effect: 0, Slight erythema (barely perceptible): 1, Well-defined erythema: 2, Moderate to severe erythema: 3, Eschar: 4

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Any test material that produces a SI of > 3 in the LLNA should be considered “positive” for contact sensitization (Kimber et al., 1994).

TREATMENT PREPARATION AND ADMINISTRATION:
- Prior to preparing the doses, the container of myclobutanil was placed in a 70°C water bath to liquefy. Once the test material melted it was maintained at that temperature throughout the dosing for the irritancy screen and LLNA (five days total).
Concentrations tested for the irritancy screen were selected based upon maximum miscibility in an appropriate LLNA vehicle while maintaining a solution suitable for application.
- The application of the test material (25 µl/ear) were made on the dorsal surface of both ears.

Positive control substance(s):

other:

Statistics:

The Stimulation Index (SI) was calculated for each mouse using the following
equation:
SI = Disintegration per minute (dpm) of individual mouse / Average dpm of the VH control mice

Means and standard deviation (SD) were generated for body weight data (absolute and gain) and the LLNA response (dpm & SI values).

Results and discussion

Positive control results:

The positive control, 30% (v/v) HCA, elicited a proliferation that was 5.3-fold greater than vehicle-treated mice.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

5.3

Variability:

SD = 2.0

Test group / Remarks:

Positive control group

Results 2

Key result

Parameter:

SI

Value:

1.6

Variability:

SD = 0.3

Test group / Remarks:

80% myclobutanil

Results 3

Key result

Parameter:

SI

Value:

1.5

Variability:

SD = 0.6

Test group / Remarks:

20% myclobutanil

Results 4

Key result

Parameter:

SI

Value:

1.1

Variability:

SD = 0.2

Test group / Remarks:

5% myclobutanil

Cellular proliferation data / Observations:

Erythema was absent in the mice treated with 5% and 20% myclobutanil, while five of the six mice treated with 80% showed slight erythema on day 6.
Body weights were unaffected in all dose groups.

Any other information on results incl. tables

Results are provided in Tables 1 and 2 below.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Topical applications with 5%, 20%, and 80% myclobutanil technical fungicide did not elicit a stimulation index that met the 3x threshold, thus indicating a lack of dermal sensitization potential in the mouse LLNA.

Executive summary:

The Local Lymph Node Assay (LLNA) was conducted with according to OECD/EC guidelines and GLP principles.

The dosing concentrations for myclobutanil in the LLNA were selected based on the results of a screening study.

In the main study, six female mice/group received 5%, 20%, or 80% myclobutanil, or vehicle (dimethyl sulfoxide (DMSO)), on days 1-3. On day 6, uptake of H-thymidine into the auricular lymph nodes draining the site of chemical application was measured five hours post-administration. Proper conduct of the LLNA was confirmed via a positive response using 30% (v/v) HCA, a moderate contact sensitizer, which elicited proliferation that was 5.3-fold greater than vehicle-treated mice.

Erythema was absent in the mice treated with 5% and 20% myclobutanil, while five of the six mice treated with 80% myclobutanil showed slight erythema on day 6. Body weights were unaffected in all dose groups. Topical application of 5%, 20%, or 80% myclobutanil elicited proliferative responses/stimulation indexes (SI) that were respectively, 1.1-, 1.5-, and 1.6- fold greater than vehicle controls.

In conclusion, myclobutanil did not demonstrate dermal sensitization potential in the mouse LLNA.