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

Endpoint summary

Description of key information

Two studies are available: an LLNA that was performed according to OECD/EC guidelines and GLP principles and a study using a modified Buehler procedure (performed before the LLNA was implemented). 

The outcome of the second study was equivocal, but the result of the LLNA indicated no skin sensitising properties of myclobutanil. 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

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)

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

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).

Positive control results:

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

Parameter:

SI

Value:

5.3

Variability:

SD = 2.0

Test group / Remarks:

Positive control group

Key result

Parameter:

SI

Value:

1.6

Variability:

SD = 0.3

Test group / Remarks:

80% myclobutanil

Key result

Parameter:

SI

Value:

1.5

Variability:

SD = 0.6

Test group / Remarks:

20% myclobutanil

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.

Results are provided in Tables 1 and 2 below.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The Local Lymph Node Assay (LLNA) was conducted 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. Myclobutanil did not demonstrate dermal sensitization potential in the mouse LLNA.

 

The delayed contact hypersensitivity potential of myclobutanil, was tested in young adult outbred Hartley guinea pigs using a modified Buehler procedure. One group of 12 guinea pigs received ten 6-hr induction doses (3 doses/week, for 3.5 weeks) of 0.4 ml of 50% W/W myclobutanil in 80% V/V aqueous ethanol. An additional group of 12 guinea pigs was treated with 1-chloro-2,4 dinitrobenzene (ONCB) at 1600 ppm in 80% V/V aqueous ethanol in the same manner and served as a positive control group. These two groups of animals and a group of 12 naive control guinea pigs (i.e. receiving no induction treatments) were challenged 2 weeks after the last induction dose. The naive control group was challenged with 0.4 ml of ONCB at 800 ppm in acetone and 50% W/W myclobutanil in acetone. The myclobutanil induced group was challenged with myclobutanil at 50% W/W in acetone and the positive control group received a challenge dose of ONCB at 800 ppm in acetone. Erythema reactions were scored 24 and 48 hr after the challenge exposure. Seven days after the primary challenge, the myclobutanil induced group was rechallenged with myclobutanil at 50% W/W in acetone and the positive control group was rechallenged with ONCB at 800 ppm in acetone. Similarly, a second naive control group of eight guinea pigs was challenged with ONCB at 800 ppm in acetone and myclobutanil at 50% W/W in acetone.

After the primary challenge, minimal to no erythema was observed in the naive control group at 24 or 48 hours following challenge with either ONCB (at 800 ppm) or myclobutanil (50% W/W). In the positive control group, 7 of 12 and 6 of 12 guinea pigs responded with erythema at 24 and 48 hours, respectively. In the myclobutanil (50% W/W) induced group, 3 of 12 and 1 of 12 animals exhibited erythema at 24 and 48 hours, respectively.

After rechallenge, no erythema was observed in the naive control group at 24 or 48 hours following challenge with either DNCB (at 800 ppm) or myclobutanil (50% W/W). In the positive control group, 6 of 12 and 5 of 12 guinea pigs responded with erythema at 24 and 48 hours, respectively. In the myclobutanil induced group, 1 of 12 and 0 of 12 guinea pigs responded with erythema at 24 and 48 hours, respectively. In the myclobutanil group, it is equivocal as to whether the erythema that was observed after challenge and rechallenge was indicative of delayed contact hypersensitivity. The low incidence of erythema responses (3 of 12 at challenge; 1 of 12 at rechallenge) could be attributed to a local primary irritation response. Overall these data are difficult to interpret and therefore are inconclusive regarding the sensitization potential of myclobutanil in guinea pigs.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data it is concluded that Myclobutanil does not meet classification criteria for skin sensitising properties according to the Classification, Labelling and Packaging (CLP) Regulation ((EC) No 1272/2008).