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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 (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October-December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD and EU guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: approx. 9 weeks old
- Weight at study initiation: 20-23 grams
- Housing: group housed in labeled Makrolon cages (MIII type) containing sterilised sawdust (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment
- Diet: free access to pelletd rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 October 2012 To: 15 October 2012
Vehicle:
methyl ethyl ketone
Remarks:
(Merck, Darmstadt, Germany)
Concentration:
10%, 25%, 40% (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 40% was the maximum concentration that could technically be applied
- Irritation: no irritation
- Lymph node proliferation response: variations in ear thickness were less than 25% from day 1 pre-dose values

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: If the results indicate a SI (Stimulation Index, ratio of the DPM/group compared to DPM/vehicle control group) ≥ 3, the test substance may be regarded as a skin sensitizer. Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3).

TREATMENT PREPARATION AND ADMINISTRATION:
Formulations (w/w) were prepared within 4 hours prior to treatment.Homogeneity was obtained to visually acceptable levels.
Induction days 1, 2 and 3: topical treatment (25 µL/ear); the concentrations were stirred prior to dosing.
Excision of the nodes day 6: injection (tail) with 20 µCi 3H-methylthymidine; after approximately 5 hours animals were killed (i.p. Euthasol®), auricular lymph node was excised.
Tissue processing for radioactivity day 6: preparation of single cell suspension of lymph node cells; DNA precipitation with TCA.
Radioactivity measurements day 7: radioactivity was determined by means of a scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
Reliability check with alpha-hexylcinnamaldehyde technical grade at concentration of 5%, 10% and 25% in acetone/olive oil (4:1 v/v). SI values were 1.7, 1.7 and 4.7 for the three concentration groups, respectively. An EC3 value of 16.5% was calculated using linear interpolation. The calculated EC3 value is in the acceptable range of 4.8% to 19.5%.
Parameter:
SI
Remarks on result:
other: The SI values calculated for the cholesterol concentrations 10%, 25% and 40% were 2.3, 2.2 and 1.3, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: mean DPM/animal values for the cholesterol concentrations 10%, 25% and 40% were 990, 959 and 548 DPM, respectively. The mean DPM/animal value for the vehicle control group was 437 DPM.

No signs of systemic toxicity and no irritation of the ears were observed.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Calculated SI values for cholesterol concentrations 10%, 25% and 40% were 2.3, 2.2 and 1.3, respectively. As Cholesterol did not elicite an SI ≥3, cholesterol is considered to be a non-skin sensitizer.
Executive summary:

Skin contact hypersensitivity to cholesterol was studied in a mouse LLNA test according to OECD guideline 429. Female CBA/J mice (5/group) were treated by application on the ear on three consecutive days with cholesterol concentrations of 10%, 25% or 40% w/w (the highest concentration that could technically be applied). A vehicle control (methyl ethyl ketone) group was included. Reliability of the test model was approved by the six-month reliability check with alpha-hexylcinnamicaldehyde (pos control).

No signs of systemic toxicity and no irritation of the ears were observed. The SI values calculated for the cholesterol concentrations 10%, 25% and 40% were 2.3, 2.2 and 1.3, respectively. As cholesterol did not elicite an SI ≥3 when tested up to the highest concentration that could technically be applied, cholesterol is considered to be a non kin sensitizer. Based on these results, cholesterol does not need to be classified and has no obligatory labelling requirements for eye irritation according to

-Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011),

-Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures

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

Skin contact hypersensitivity to cholesterol was studied in a mouse LLNA test according to OECD guideline 429. Female CBA/J mice (5/group) were treated by application on the ear on three consecutive days with cholesterol concentrations of 10%, 25% or 40% w/w (the highest concentration that could technically be applied). A vehicle control (methyl ethyl alcohol) group was included. Reliability of the test model was approved by the six-month reliability check with alpha-hexylcinnamicaldehyde (pos control).

No signs of systemic toxicity and no irritation of the ears were observed. The SI values calculated for the cholesterol concentrations 10%, 25% and 40% were 2.3, 2.2 and 1.3, respectively. As cholesterol did not elicite an SI ≥3 when tested up to the highest concentration that could technically be applied, cholesterol is considered to be a non kin sensitizer. Based on these results, cholesterol does not need to be classified and has no obligatory labelling requirements for eye irritation according to

-Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011),

-Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures


Migrated from Short description of key information:
For determination of skin sensitization, a reliable LLNA study with cholesterol in mouse is available.

Justification for selection of skin sensitisation endpoint:
Reliable study, performed according to OECD and EC guidelines and according to GLP principles.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

As cholesterol did not elicite an SI ≥3 when tested up to the highest concentration that could technically be applied in a reliable LLNA study in mice, cholesterol is considered to be a non skin sensitizer. Based on these results, cholesterol does not need to be classified and has no obligatory labelling requirements for skin sensitisation according to

-Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011),

-Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures

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