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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Good publication according to OECD, EU , US guide lines.
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
no
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Winkelmann
- Age at study initiation:6-12 weeks
- Weight at study initiation:300–500 g
- Housing:The guinea pigs were housed in groups in Terluran cages on Altromin saw fiber bedding or Lignocel bedding. The animals were barrier-maintained (semi-barrier) in an air conditioned room.
- Diet:Altromin 3122 maintenance diet for guinea pigs or ssniff Ms-H, 4mm V2233-000 complete diet for guinea pigs, rich in crude fiber, totally-pathogen-free), ad libitum
- Water : tap water, ad libitum
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 3 °C
- Humidity (%):55 ± 10%
- Air changes (per hr):10 air change per hour
- Photoperiod (hrs dark / hrs light):12 hours cycle dark/light

Route:
intradermal
Vehicle:
physiological saline
Remarks:
Isotonic saline (NaCl 0.9%)
Concentration / amount:
Intradermal induction
0.1 ml each of the test compound in vehicle, the test compound in a 1:1 (v/v) mixture of FCA/physiological saline and a 1:1 (v/v) mixture of FCA/physiological
saline without test compound.

Topical induction
0.5 ml of Squalene

Change:
0.5 g of squalene
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Remarks:
Isotonic saline (NaCl 0.9%)
Concentration / amount:
Intradermal induction
0.1 ml each of the test compound in vehicle, the test compound in a 1:1 (v/v) mixture of FCA/physiological saline and a 1:1 (v/v) mixture of FCA/physiological
saline without test compound.

Topical induction
0.5 ml of Squalene

Change:
0.5 g of squalene
No. of animals per dose:
10 females x dose as test
5 females x dose as control
Details on study design:
RANGE FINDING TESTS:A range finding for the test concentrations was performed in a preliminary experiment assessing the irritant effects of the test compounds after intradermal and topical application.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures:2, an intradermal injection (day 0) and a topical application (day 7). Approximately 24 h before the topical induction
treatment (day 6) the test animals received a topical application of 0.5 ml of 10% sodium lauryl sulphate in Vaseline in order to create a mild local
skin irritation.
- Test groups:10 females
- Control group:5 females
- Site:no data, the intradermal induction comprised three pairs of injections.
- Frequency of applications:day 0, day 7
- Concentrations:The intradermal induction comprised three pairs of injections of 0.1 ml each of the test compound in vehicle, the test compound in a 1:1 (v/v) mixture of FCA/physiological saline and a 1:1 (v/v) mixture of FCA/physiological saline without test compound. The control animals received applications without test substance.

B. CHALLENGE EXPOSURE
- No. of exposures:2, topical challenge treatment on day 20. In addition to the requirements of the OECD guideline, a second topical challenge was performed on day 28 in all experiments.
- Day(s) of challenge:day 20 and 28.
- Exposure period: 24 h
- Test groups:10 females
- Control group:5 females. Control animals received the vehicle without test compound.
- Site:no data
- Concentrations:0.5 g
- Evaluation (hr after challenge):Approximately 21 h after removal of the challenge patch, the skin area was cleaned and cleared of hair. About 24 an d 48 h after removal of the patch, the skin reaction was observed and recorded. An additional reading of skin reactions was performed at 72 h after patch removal.

Positive control substance(s):
no
Reading:
1st reading
Hours after challenge:
24
Group:
test group
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
isolated grade 1 skin reactions
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: isolated grade 1 skin reactions.

No signs of systemic toxicity were observed for any of the test substances in any of the animals. For squalene, one animal showed a grade 1 skin reaction at all reading time points after the first challenge as well as at all readings after the re-challenge. Thus, this animal clearly was sensitized to the test substance. Another two animals showed a skin reaction only at the 72 h reading after the first challenge. Upon re-challenge, the reaction was reproducible in only one of these two animals. Conclusively, two animals were considered sensitized to squalene.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The positive response was below 30% of test group therefore Squalene is considered as non sensitizer for guinea pig.
Executive summary:

Squalene as been tested for skin sensitisation in accordance with OECD, EU and US EPA guidelines. A range finding for the test concentrations was performed in a preliminary experiment assessing the irritant effects of the test compounds after intradermal and topical application. Each test was performed with 10 test and five control animals because this number of animals has been reported as sufficient for the GPMT and is, in general, accepted by authorities. The positive response was below 30% of test group therefore Squalene is considered as non sensitizer for guinea pig.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a comprehensive study performed in the framework of official validation of LLNA method (Kreiling 2008), Squalene was tested both with LLNA and in vivo, on guinea pig.

According to EC guideline B.42 (LLNA) The test compound were assayed at three concentrations. If technically possible, the vehicle was AOO (3 + 1(v/v) acetone/olive oil). In the LLNA, no signs of systemic toxic effects were observed with test substance. No clinical signs of local irritation were observed by visual inspection of the ears. Similarly, no significant ear swelling response was measured at any of the test concentrations.

Squalene give clear positive results in the LLNA with the highest SI value.

Squalene has been tested for skin sensitisation on guinea pig in accordance with OECD, EU and US EPA guidelines. A range finding for the test concentrations was performed in a preliminary experiment assessing the irritant effects of the test compounds after intradermal and topical application. Each test was performed with 10 test and five control animals because this number of animals has been reported as sufficient for the GPMT and is, in general, accepted by authorities. The positive response was below 30% of test group therefore Squalene is considered as non sensitizer for guinea pig.

The substance was previously tested again on guinea Pig (Ben´ıšek Z., 2004) with negative results.

Squalene has been evaluated as a non sensitizer and it has been inserted in the substance category that restitutes a false positive in LLNA.


Migrated from Short description of key information:
Not sensitizing

Justification for selection of skin sensitisation endpoint:
In vivo study

Justification for classification or non-classification