Cholesterol

Skin sensitisation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

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)

Test material

Test material information

Constituent 1

Reference substance name:

Cholesterol

EC Number:

200-353-2

EC Name:

Cholesterol

Cas Number:

57-88-5

Molecular formula:

C27H46O

IUPAC Name:

cholest-5-en-3-ol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): cholesterol
- Physical state: white powder
-storage conditions: at room temperature in the dark

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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)

Results and discussion

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%.

In vivo (LLNA)

Results

Results 1

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.

Results 2

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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