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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2012-09-26 to 2012-10-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(5'-((5S)-5-(3,5-Dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-3'-H-spiro(azetidine-3,1'-(2)benzofuran)-1-yl)-2-(methylsulfonyl)ethanone
Cas Number:
1398609-39-6
Molecular formula:
C23H18Cl2F4N2O5S
IUPAC Name:
1-(5'-((5S)-5-(3,5-Dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-3'-H-spiro(azetidine-3,1'-(2)benzofuran)-1-yl)-2-(methylsulfonyl)ethanone
Specific details on test material used for the study:
Lot No: PF-06450567-00-0022
Purity: Not provided

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley; Indianapolis, IN
- Females nulliparous and non-pregnant: yes
- Age at study initiation: ca. 10 weeks
- Weight at study initiation: 17.9 - 22.9 g (range-finding); 19.8 - 25.3 g (main test)
- Housing: Polycarbonate boxes with bedding, 1-5 per cage
- Diet: PMI Feeds, available ad libitum
- Water: Municipal water, available ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 42-91
- Air changes (per hr): 10+ air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle
- IN-LIFE DATES: From: 10 Oct 12 To: 15 Oct 12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.5%, 1% and 2.5% v/v in 80% acetone:20% olive oil
No. of animals per dose:
5 females per dose
Details on study design:
PRE-SCREEN TESTS:
Healthy mice were released from quarantine prior to testing. Five females/group were selected for irritation screening to determine the three consecutive concentrations for the main test. Tested were 100% undiluted, and 50, 25 and 5% v/v dilutions of test item in acetone:olive oil. All Test group animals dosed higher than 5% died during the screening; therefore, lower doses were selected for the main test.

MAIN STUDY
- Test Item Preparation and Administration:
Five females were selected for each of three Test groups (Groups I - III). On Days 1, 2 and 3, each Test animal in its group received an open application of 25 μL of an appropriate dilution (0.5, 1 or 2.5%) of the test item to the dorsum of both ears. The Vehicle Control group (5 females) was treated in the same way as test animals, but with vehicle alone (acetone:olive oil) instead of test item dilution. The Positive Control group (5 females) was treated with 80% alpha-hexylcinnamaldehyde in acetone:olive oil. All Test and Control animals were given a two-day rest period on Days 4 and 5.
- Injection of Tritiated Methyl-Thymidine
On Day 6 of the study, all Test and Control animals were injected in the tail vein with 250 μL of 0.01 M phosphate-buffered saline, pH 7.4 at 25℃ per manufacturer, containing 20 μCi of [methyl-3H] Thymidine. Five hours after the injection, the animals were sacrificed with an overdose of CO2, the draining auricular lymph nodes were excised and pairs from each individual animal were processed.
- Body Weights and Observations:
Individual body weights were recorded on Day 1 prior to dosing, and Day 6, prior to injection. All Test and Control animals were observed daily for clinical signs of toxicity and any signs of excessive irritation at the test site.
- Criteria used to consider a positive response: stimulation index >3
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Stimulation index: 4.9, positive

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.5
Test group / Remarks:
Test Group III; Test Item Concentration: 2.5%
Parameter:
SI
Value:
2.3
Test group / Remarks:
Test Group II; Test Item Concentration: 1%
Parameter:
SI
Value:
1.2
Test group / Remarks:
Test Group I; Test Item Concentration: 0.5%
Cellular proliferation data / Observations:
Four animals in Test Group I and three in Test Group II lost/failed to gain weight during the study; two animals in each Control group also lost weight.
Two Test Group III animals were found dead on Day 4; otherwise, all animals appeared normal for the duration of the study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item produced a stimulation index of < 3 in all groups of Test animals, and is not therefore considered a sensitizer (defined as producing a positive response).
Executive summary:

A skin sensitization study was conducted on 3 groups of 5 female mice to determine if test item possesses a significant potential to cause skin sensitization according to OECD 429. Five females were assigned to each of three groups, designated Groups I - III. The Test groups were treated with an appropriate dilution (0.5, 1 or 2.5%) in acetone:olive oil vehicle. Each animal received 25 μL to the dorsum of each ear. The animals were treated once daily for three days. After a two-day rest period, all animals were injected with tritiated methyl-thymidine in the tail vein. Five hours later, the animals were sacrificed, and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A Vehicle Control group of five females was run concurrently, treated in the same manner with vehicle only instead of test item dilution. A Positive Control group of five females was also run concurrently, treated with 80% alpha-hexylcinnamaldehyde in acetone:olive oil.


The test item produced a stimulation index of < 3 in all groups of Test animals, and is not therefore considered a sensitizer (defined as producing a positive response).