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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:
From August 17, 2011 to September 22, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 406 and OPPTS 870.2600, in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
Buehler test

Test material

Constituent 1
Chemical structure
Reference substance name:
3-phenyl-5-(2-thienyl)-1,2,4-oxadiazole
EC Number:
810-533-8
Cas Number:
330459-31-9
Molecular formula:
C12H8N2OS
IUPAC Name:
3-phenyl-5-(2-thienyl)-1,2,4-oxadiazole
Test material form:
other: Cream coloured solid
Details on test material:
- Name of test material (as cited in study report): MON 102100
- Prior to use, the test substance was ground in a coffee mill (Cuisinart, Model DCG-20N).

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Received from Elm Hill Breeding Labs on July 20 and August 10, 2011 (preliminary irritation group) and August 10, 2011 (test and naive control groups).
- Age at study initiation: Young adult
- Weight at study initiation: 366-473 g
- Housing: The animals were group housed in suspended stainless steel caging with mesh floors or plastic perforated bottom caging which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Litter paper was placed beneath the cage and was changed at least three times per week.
- Animal room temperature: 19-23°C
- Humidity: 57-76%. The humidity was above the targeted upper limit for 12 d during the study. A portable dehumidifier was used to lower the humidity levels during this time.
- Animal room air changes/h: 13, airflow measurements are evaluated regularly and the records are kept on file at PSL.
- Acclimation Period: 13-34 d
- Photoperiod (h dark / h light): 12 h light/ 12 h dark cycle
- Food: Harlan Teklad Global Guinea Pig Diet® #2040. A designated amount of the diet was available to each guinea pig (approximately 20 grams/day).
- Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system.

Analyses of the food and water were conducted regularly. There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study.

Identification
- Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal.
- Animal: Each guinea pig was marked with a colour code and given a sequential animal number assigned to study 32890, which constituted unique identification

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
(2%)
Concentration / amount:
Preliminary irritation testing: 4, 9, 18, 35, 53 and 70%

Main test:
Induction: 70% w/w
Challenge: 18% w/w
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
(2%)
Concentration / amount:
Preliminary irritation testing: 4, 9, 18, 35, 53 and 70%

Main test:
Induction: 70% w/w
Challenge: 18% w/w
No. of animals per dose:
5 (preliminary irritation testing); 20 (test group); 10 (naive control group).
Details on study design:
PRIMARY IRRITATION STUDY
A group of animals were used to determine the highest non-irritating concentration (HNIC) of the test substance prior to the challenge dose. The fur was removed by clipping the dorsal area and flanks of each guinea pig. This area was divided into four test sites (two sites on each side of the midline) on each animal. The test substance was mixed with a 2% w/v solution of CMC in distilled water to yield w/w concentrations of 70% (0.4g), 53% (0.4g), 35% (0.4 mL), 18% (0.4 mL), 9% (0.4 mL) and 4% (0.4 mL). Each concentration was applied (0.4 g or mL) to a test site using an occlusive 25 mm Hill Top Chamber. The sites were wrapped with non-allergenic Durapore adhesive tape. After 6 h of exposure, the chambers were removed and the test sites wiped free with a mild soap solution and a water rinse using clean towels to remove any residual test substance. Approximately 24 h after application, each site was evaluated for local reactions (erythema) according to below scoring system.
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 an 18% w/w mixture in a 2% w/v solution of CMC in distilled water.

SCORING SYSTEM:
0 - no reaction
0.5 - very faint erythema, usually non-confluent*
1 - faint erythema, usually confluent
2 - moderate erythema
3 - severe erythema with or without oedema
*Very faint erythema is not considered a positive reaction.

MAIN STUDY

PREPARATION AND SELECTION OF ANIMALS
On the day before initiation, the fur of a group of animals was removed by clipping the dorsal area and flanks. After clipping and prior to initiation, the animals were weighed and the skin was checked for any abnormalities. Only healthy naive animals (not previously tested) without pre-existing skin irritation were selected for test. Animals were re-clipped prior to each dose.

A. INDUCTION EXPOSURE
Once each week for three weeks, 0.4 g of a 70% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water was applied to the left side of each test animal using an occlusive 25 mm Hill Top Chamber. The chambers were secured in place and wrapped with non-allergenic Durapore adhesive tape to avoid dislocation of the chambers and to minimize loss of the test substance. After the 6 h exposure period, the chambers were removed and the test sites wiped free with a mild soap solution and a water rinse using clean towels to remove any residual test substance. Approximately 24 and 48 h after each induction application, readings were made of local reactions (erythema).

B. CHALLENGE EXPOSURE
28 d after the first induction dose, 0.4 mL of an 18% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water (HNIC) was applied to a naive site on the right side of each animal as a challenge dose, using the procedures described above. These sites were evaluated for a sensitization response (erythema) approximately 24 and 48 h after the challenge application. In addition to the test animals, 10 guinea pigs from the same shipment were maintained under identical environmental conditions and were treated with the HNIC of the test substance at challenge only. These animals constituted the "naive control" group.

- Individual body weights of the animals were recorded prior to initiation and again on the day after challenge.
Positive control substance(s):
yes
Remarks:
75% w/w Alpha-Hexylcinnamaldehyde (HCA) in mineral oil; Historical Positive Control Validation Study

Results and discussion

Positive control results:
Induction phase:
Test animals (100% HCA): Very faint erythema (0.5) was noted at approximately half of the positive control dose sites during the induction phase.

Challenge phase:
Historical positive control animals (75% w/w mixture of HCA in mineral oil): Three of ten positive control animals exhibited signs of a sensitization response (faint erythema [1]) 24 h after challenge. Similar indications persisted at one of these sites through 48 h. Very faint erythema (0.5) was noted for three other sites following challenge.

Naive Control Animals (75% w/w mixture of HCA in mineral oil): Very faint erythema (0.5) was noted at one of five naive control sites 24 h after challenge. Irritation cleared from this site by 48 h.

The positive response observed in the historical positive control study validates the test system used in this study.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
18% w/w
No. with + reactions:
4
Total no. in group:
19
Clinical observations:
Very faint erythema (0.5)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 18% w/w. No with. + reactions: 4.0. Total no. in groups: 19.0. Clinical observations: Very faint erythema (0.5).
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
18% w/w
No. with + reactions:
1
Total no. in group:
19
Clinical observations:
Very faint erythema (0.5)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 18% w/w. No with. + reactions: 1.0. Total no. in groups: 19.0. Clinical observations: Very faint erythema (0.5).

Any other information on results incl. tables

- One test animal was euthanized for humane reasons due to imminent death following the challenge application. Gross necropsy of the euthanized animal revealed a blood filled abdominal cavity and distention of the stomach and intestines.

Induction phase

Very faint to faint erythema (0.5-1) was noted for all test sites during the induction phase.

Challenge phase

Test animals: Very faint erythema (0.5) was noted for four of nineteen test sites 24 h after challenge. Irritation persisted at one of these sites through 48 h.

Naive control animals: Very faint erythema (0.5) was noted for four of ten naive control sites 24 h after challenge. Irritation cleared from the affected site by 48 h.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the study conditions, the test substance was not considered to be a skin sensitiser.
Executive summary:

A skin sensitisation test was performed on guinea pigs to determine the sensitisation potential of the test substance using the Buehler Method according to OECD Guideline 406 and OPPTS 870.2600, in compliance with GLP. A 70% w/w mixture of the test substance in a 2% w/v solution of carboxymethylcellulose (CMC) in distilled water was topically applied to 20 healthy test guinea pigs, once each week for a three week induction period. 28 d 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 an 18% w/w mixture in a 2% w/v solution of CMC in distilled water) was applied to a naive site on each guinea pig. A naive control group (10 animals) was maintained under the same environmental conditions and treated with the test substance at challenge only. Approximately 24 and 48 h after each induction and challenge dose, the animals were scored for erythema. Very faint to faint erythema (0.5-1) was noted for all test sites during the induction phase. Very faint erythema (0.5) was noted for four of nineteen test sites 24 h after challenge and irritation persisted at one of these sites through 48 h. Very faint erythema (0.5) was noted for four of ten naive control sites 24 h after challenge. However, irritation cleared from the affected site by 48 h. The positive response observed in the historical positive control validation study with alpha-hexylcinnamaldehyde (HCA) validates the test system used in this study. Based on the study results, the test substance was not considered to be a skin sensitiser (Durando J, 2011e).