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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-March-1994 to 21-April-1994.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
June 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed before the LLNA was available.

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(4-chlorophenyl)-2-phenyl-2-[(1H-1,2,4-triazol-1-yl)methyl]butanenitrile
EC Number:
406-140-2
EC Name:
4-(4-chlorophenyl)-2-phenyl-2-[(1H-1,2,4-triazol-1-yl)methyl]butanenitrile
Cas Number:
114369-43-6
Molecular formula:
C19H17ClN4
IUPAC Name:
4-(4-chlorophenyl)-2-phenyl-2-[(1H-1,2,4-triazol-1-yl)methyl]butanenitrile
Test material form:
solid: particulate/powder
Remarks:
Off white powder.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
The animals were supplied by Harlan Sprague Dawley, Inc., Indianapolis (USA). The animals weighed between 346 and 617 grams at the start of the study. Prior to use, all animals were acclimated for at least eight days. Animals were individually housed in wire mesh suspension' cages. Teklad Guinea Pig Diet and tap water were supplied ad libitum during both acclimation and test periods. There were no contaminants in either the feed or the water that were expected to affect the outcome of this study.
Environmental conditions in the animal room were maintained at a temperature of 63.40 to 73.2° F. and a relative humidity of 30.3% to 85.3%. The animals were maintained on a 12-hour light/12-hour dark cycle. The food is routinely analyzed by the manufacture for nutritional components and environmental contaminants. Samples of the water are analyzed for total dissolved solids, hardness, and specified microbiological content and for selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
acetone/olive oil (4:l v/v)
Concentration / amount:
10% / 0.1 mL
Day(s)/duration:
Day 0
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, semiocclusive
Vehicle:
acetone/olive oil (4:l v/v)
Concentration / amount:
0.8 ml 25% w.v.
Day(s)/duration:
Day 7
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
acetone/olive oil (4:l v/v)
Concentration / amount:
0.4 mL as 10% w/v applied to a patch system on one site.
Day(s)/duration:
Day 21
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Primary Irritation: 5 males, 5 females
Test: 9 males, 10 females
Vehicle control: 5 males, 5 females
Naive controls: 10 males, 9 females
Details on study design:
Irritation screening (pilots):
The hair on the back of each animal was removed using a small animal clipper the day prior to test material application. This injection irritation screen utilized two animals, each receiving eight injections. These animals received eight concentrations of Fenbuconazole as 25%, 10%, 5%, 2.5%, 1%, 0.5%, and 0.25% w/v formulations in acetone and undiluted acetone. A 0.1 ml amount of the appropriate test material formulation was injected intradermally. Injections were given in two rows one on either side of the dorsal midline.

Topical application study phase: Two sets of four animals were used for this phase of the study. The hair on the back of each animal was removed using a small animal clipper the day prior to test material application. The first set of four animals received four concentrations per animal, Fenbuconazole as 50%, 25%, 10%, and 5% w/v formulations in acetone. The second set of four animals received Fenbuconazole as 2.5%, 1%, 0.5%, and 0.25% w/v formulations in acetone.

A 0.4 ml amount of the appropriate formulation was applied onto a 20 mm x 20 mm Webril pad. The patches were applied to the clipped area as quickly as possible. The patches were secured over the clipped surface using an appropriate width of Elastoplast" elastic adhesive bandage wound around the torso of the animal until overlapping. The position of the different concentrations were varied to adjust for possible site-ta-site variation in response. The patch sites utilized Sites 1 - 4 as indicated in the above diagram. The wrappings and patches were removed approximately twenty-four (24) hours later. The day following patch removal, the application sites were depilated and scored.

Observations: Twenty-one (21) hours following topical pilot, primary challenge, or rechallenge patch removal, all animals were depilated with a commercial depilatory. The depilatory was placed on the application sites and surrounding area. The depilatory was left on for no more than fifteen minutes. The depilatory was thoroughly removed with warm, running water. The animals were then dried with a towel and returned to their cages.

Three hours later, (24 hours following challenge patch removal), the test sites were graded using the following scale:
0 = no perceptible reaction or any occurrence of slight, ill-defined erythema
1 = slight but confluent, or moderate patchy erythema
2 = moderate erythema
3 = severe erythema with or without edema
The grading was repeated the following day, 48 hours following challenge patch removal.
The animals were observed daily for any clinical abnormalities. There were no clinical abnormalities noted during the observation period.
Challenge controls:
Primary Challenge Phase; Topical Application:
On Day 20 of the study, both the lower left and the lower right flanks of each animal were clipped using a small animal clipper. On Day 21 of the study, test animals and vehicle control animals received a 0.4 mL of Fenbuconazole as a 10% w/v formulation in acetone applied to a patch system in one site and a 0.4 mL amount of undiluted acetone applied to another patch system at another site.

At this same time, the naive control animals received a 0.4 mL amount of Febuconazole as a 10% w/v formulation in acetone applied to a patch system and a 0.4 mL amount of undiluted acetone applied to another patch system. The two patches were rotated between sites.

Each patch was secured over the clipped area using an appropriate width of elastic adhesive bandage which was wound around the torso of each animal until overlapping. The wrappings and patches were removed approximately twenty-four hours later. The day following patch removal, the application sites were depilated and scored according to the scale described
under the "Observations" section.


Rechallenge Phase: Topical Application:
On Day 33 of the study, both the middle left and the middle right flanks of each animal were clipped using a small animal clipper. On Day 34 of the study, test animals and vehicle control animals received a 0.4 mL amount of Fenbuconazole as a 10% w/v formulation in diethyl phthalate applied to a patch system at Site 7 and a 0.4 mL amount of undiluted diethyl phthalate applied to another site.
Positive control substance(s):
yes
Remarks:
alpha - Hexylcinnamaldehyde (historical control data)

Results and discussion

Positive control results:
a-Hexylcinnamaldehyde as a 5% w/v formulation in acetone exhibited a sensitization rate of 100%.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
induction: 0%; challenge: 10% in aceton
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%; Challenge: 10% in aceton
No. with + reactions:
2
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0%; Challenge: 10% w/v in Diethyl phthalate.
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%; Challenge: 10% w/v in Diethyl phthalate.
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Induction: 10%; Challenge: 10% w/v in Acetone.
No. with + reactions:
2
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Induction: 10%; Challenge: 10% w/v in Acetone.
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
Induction: 10%; Challenge: 10% w/v in Diethyl phthalate.
No. with + reactions:
3
Total no. in group:
19
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
Induction: 10%; Challenge: 10% w/v in Diethyl phthalate.
No. with + reactions:
2
Total no. in group:
19
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
85%
No. with + reactions:
10
Total no. in group:
10

Any other information on results incl. tables

One naive control animal died on Day 31 of the study. Its participation on this study ended with the primary challenge scoring. A necropsy was performed on this animal, and the findings included pale lungs, mottled liver, pale and congested kidneys, and the stomach was distended with gas and contained a small amount of green food-like material. There were no other gross pathological finding observed.
One test animal animal died on Day 34 of the study. Its participation on this study ended with the day of rechallenge application. A necropsy was performed on this animal, and the findings included pale lungs, pale and congested kidneys, and the stomach was distended with gas, and slight post-mortem autolysis was evident. There were no other gross pathological finding observed.


There were no clinical abnormalities noted during the observation period. The animals weighed between 346 and 617 grams at the start of the study. The animals showed an increase in weight throughout the study, weighing between 391 and 781 grams at the end of the study.


 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of a delayed contact hypersensitivity study in guinea pigs, it was concluded that the test item was not a skin sensitiser.