Cholesterol

Endpoint summary

Description of key information

For determination of skin irritation/corrosion, a reliable in vitro skin irritation test with cholesterol using a human skin model is available. In addition, an acute dermal toxicity study with no dermal effects is available.
For determination of eye irritation, a reliable acute eye irritation/corrosion study with cholesterol in the rabbit is available.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July-October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: human epidermis model (human-derived epidermal keratinocytes)

Details on test animals or test system and environmental conditions:

test system:
three-dimensional human epidermis model, consisting of adult human-derived epidermal keratinocytes, seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, resulting in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

source:
SkinEthic Laboratories, Lyon, France.

Type of coverage:

other: in vitro system

Amount / concentration applied:

TEST MATERIAL
- Amount applied (volume or weight with unit): 10.2 to 11.5 mg cholesterol.

Duration of treatment / exposure:

15 minutes at room temperature, post-incubation for 42 hours at 37°C.

Details on study design:

Cytotoxicity (irritancy) is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of treatment.

Irritation / corrosion parameter:

other: other: cytotoxicity

Value:

102

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes exposure. Reversibility: other: not relevant. Remarks: in vitro study. (migrated information)

Irritant / corrosive response data:

mean tissue viability:
negative control: 100%
cholesterol: 102%
pos control: 5%

acceptability of the assay.

the test is considered acceptable if it meets the following criteria:

-the absolute mean optical density at 570 nm of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the SD value of the % viability should be ≤18

-the mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the SD value of the % should be ≤18

-the SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18

A test substance is considered irritant in the skin irritation test if:

The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hors of post-incubation is ≤50% of the mean viability of the negative controls.

A test substance is concidered non-irritant in the skin irritation test if:

The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation >50% of the mean viability of teh negative controls.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Cholesterol is non-irritant in a valid in vitro skin irritation test.

Executive summary:

Cholesterol was tested for skin irritaing potential with a human three dimensional epidermal model (EPISKIN-Small Model). 10.2 to 11.5 mg cholesterol was applied directly on top of the skin tissue for 15 minutes, and after a 42 hours post-incubation period the cytotoxic effect (as indicator for irritancy) was determined by means of the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT. Mean relative tissue viability above 50% is considered non-irritant. Adequate positive and negative controls were included. The relative mean tissue viability after treatment with cholesterol compared to the negative control tissue was 102%. Based on these results, cholesterol is considered non-irritant in this in vitro test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

june 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD and EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF guidelines (2000); including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: at least 6 weeks
- Weight at study initiation: 1733 to 2267 grams
- Housing: individually inlabeled cages with perforated floors (Ebeco, germany) and shelters (Ebeco, Germany)
- Diet: ad libitum pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad, Italy); Hay (Technilab-BMI BV, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, The Netherlands).
- 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): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 June 2012 To: 22 June 2012

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount applied (volume or weight with unit): on average 21.6 mg (range 21.4-21.7 mg), a volume of approximately 0.1 mL

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

1, 24, 48 and 72 hours after instillation

Number of animals or in vitro replicates:

3 males

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: no

SCORING SYSTEM: according to guideline (scoring system presented in OECD 405

TOOL USED TO ASSESS SCORE: fluorescein (2% solution)

OTHER OBSERVATIONS: mortality/viability: twice daily; toxicity: at least once daily; body weight: before exposure and after the final observation.

Irritation parameter:

cornea opacity score

Basis:

other: mean of 3 animals

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

other: mean of 3 animals

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

other: mean of 3 animals

Time point:

other: 24, 48 and 72 hours

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

other: mean of 3 animals

Time point:

other: 24, 48 and 72 hours

Score:

0.1

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Instillation of approximately 0.1 mL cholesterol (approximately 22 mg) into one eye of each of three rabbits resulted in irritation of the conjunctivae (redness, chemosis and discharge). Irridial irritation grade 1 was observed for one animal at 1 hour after instillation. The irritation was fully reversible within 24, 48 or 72 hours in the respective animals. No corneal opacity nor corneal epithelial damage has been observed.
Remnants of the substance were present in the eyes of all animals at approximately 1 hour after instilation.

Other effects:

No mortality and no symptoms of systemic toxicity were observed during the test period.

Mean value eye irritation scores        (Mean 24, 48 and 72 hours )
			conjunctivae
Animal	corneal opacity	iris	redness	chemosis
1	0.0	0.0	0.0	0.0
2	0.0	0.0	0.3	0.0
3	0.0	0.0	0.7	0.3

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the results with the eye irritation study, cholesterol is considered non-irritating to eyes. As a result, cholesterol does not have to be classified and has no obligatory labelling requirements for eye irritation acoording to GHS and EU Regulation 1272/2008.

Executive summary:

An eye irritation study with cholestrol was performed according to OECD and EC guidelines. Three rabbits received approximately 0.1 mL (approximately 22 mg) cholesterol in one eye. Irritation of the eye was scored after 1, 24, 48 and 72 hours after instillation of the substance in the eye.

Irritation of the conjunctivae (redness, chemosis and discharge) was observed. Iridial irritation grade 1 was observed for one animal at 1 hour after instillation. The irritation was fully reversible within 24, 48 or 72 hours in the respective animals. No corneal opacity nor corneal epithelial damage has been observed. Remnants of the substance were present in the eyes of all animals at approximately 1 hour after instilation. Based on these results, Cholesterol needs not 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 irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Cholesterol was tested for skin irritaing potential with a human three dimensional epidermal model (EPISKIN-Small Model). 10.2 to 11.5 mg cholesterol was applied directly on top of the skin tissue for 15 minutes, and after a 42 hours post-incubation period cytotoxic effect (as indicator for irritancy) was determined by means of the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT. Mean relative tissue viability above 50% is considered non-irritant. Adequate positive and negative controls were included. The relative mean tissue viability after treatment with cholesterol compared to the negative control tissue was 102%. Based on these results, cholesterol is considered non-irritant in this in vitro test. Moreover, in the reliable available acute dermal toxicity study, no dermal effects were observed up to and including 2000 mg/kg bw.

Irritation of the conjunctivae (redness, chemosis and discharge) was observed. Irridial irritation grade 1 was observed for one animal at 1 hour after instillation. The irritation was fully reversible within 24, 48 or 72 hours in the respective animals. No corneal opacity nor corneal epithelial damage has been observed. Remnants of the substance were present in the eyes of all animals at approximately 1 hour after instilation. Based on these results, cholesterol is considered non-irritating to the eyes.

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

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

Justification for classification or non-classification

Based on the absence of severe and/or irrevesible irritating effects on the skin and the eyes, cholesterol does not need to be classified and has no obligatory labelling requirements for skin irritation and 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