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Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-10-12 to 12-11-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline and GLP compliant

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 439 Guideline For The Testing of Chemicals: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Version / remarks:
2010
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one
EC Number:
251-020-3
EC Name:
[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one
Cas Number:
32388-55-9
Molecular formula:
C17H26O
IUPAC Name:
[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Methyl Cedryl Ketone
- Physical state: Liquid
- Lot/batch No.:0100172226
- Expiration date of the lot/batch:23-07-2014
- Storage condition of test material:Roomtemperature (ca 20°C) in the dark

Test animals

Details on test animals or test system and environmental conditions:
EPISKIN™ Skin Irritation Test 15 Min-42 Hours using human epidermis skin constructs with a functional multi-layered stratum corneum was used. The epidermis surface area supplied is 0.38cm2. The kit was supplied by SkinEthic Laboratories, Lyon, France.

The Technical Data, Safety Sheet and Certificate of Analysis for the EPISKIN construct kit that provides information on performance (viability, barrier function and morphology) is attached to the study record as Annex I.

Batch number: 12-EKIN-041; Passage number: Second. The kit expiry date was: 12/11/12
On receipt of the kit, the inserts with tissues on agarose were stored at room temperature until use.

Reduction of MTT by test substance: The MTT reducing capability of the test substance was investigated by mixing approximately 10 µL ofthe test substance with2 mL of 0.3 mg/mL MTT solution in duplicate. A control of 10 µL ofdistilled water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.

Check for colouring potential of test substance: The test substance was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). The test substance (10 µL) was mixed with 90 µL of distilled water in a transparent container. 100 µL of distilled water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the
colour of the solution was assessed by eye.

Test substance pH: An estimate of the pH of the test substance as a 10% (v/v) solution in distilled water was determined using pH indicator paper and recorded.

Test system

Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL of the test substance were applied to each of triplicate tissues.

Positive controls: 10 µL to each of triplicate tissues (5% Sodium Dodecyl Sulphate (SDS) in distilled water)
Negative controls: 10 µL to each of triplicate tissues (Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium.
Duration of treatment / exposure:
15 ± 0.5 minutes
Details on study design:
Replicates: Triplicate tissues of human epidermis skin were dosed with the test substance, negative and positive control.

Positive controls: 5% Sodium Dodecyl Sulphate (SDS) in distilled water
Negative controls: Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS
- Time after start of exposure: 15 ± 0.5 minutes

After incubation for 42 ± 1 hr at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, each tissue was incubated with 2 mL of 0.3 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The
epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro tube and vortexed with 500 µL of acidic isopropanol (0.04 N HCl final concentration). The tissues were extracted by storing at 2-8 ºC, protected fromlight, for 48 to 70 hours. The absorbance of the extractant was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

SCORING SYSTEM: The mean Optical Density (OD) for the six replicate blanks was subtracted from the individual substance and control tissues OD. The mean absorbance of the tri plicate negative control values should be ≥0.6 and ≤1.5. The Standard Deviation (SD) value ofthe % viability should be <18. The OD of the positive control is an indicator of the sensit ivity of the tissues. The mean viability shoul d be ≤40% ofthe negative control and the SD <18%. If the mean tissue viabilit y was equal to or less than 50% of the negative control value, the sample was classed as Irritant R38 (EU classification) or Category2 (GHS classification).

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
100
Remarks on result:
other:
Remarks:
Basis: mean negative control (Tissue 1,2,3). Remarks: Tissue viability as percentage of mean OD negative control . (migrated information)
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
24.3
Remarks on result:
other:
Remarks:
Basis: mean positive control (Tissue 1,2,3). Remarks: Tissue viability as percentage of mean OD negative control . (migrated information)
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
76.2
Remarks on result:
other:
Remarks:
Basis: mean Methyl Cedryl Ketone (Tissue 1,2,3). Remarks: Tissue viability as percentage of mean OD negative control . (migrated information)
Other effects / acceptance of results:
Individual tissue data (a,b,c) is presented below for negative and positive control and test substance (Table 1).

There was no change in the test substance, Methyl Cedryl Ketone/MTT solution or the water control/MTT solution after three hours incubation in the dark 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The test substance had not interacted with the MTT.

The test substance, Methyl Cedryl Ketone/water solution and water control were colourless after the 15 minute shaking period. The test substance, Methyl Cedryl Ketone, had not shown any potential for colouring water.

The pH of the test substance, Methyl Cedryl Ketone, as a 10% v/v solution in purified water, measured using pH indicator paper, was approximately 7.0.

Any other information on results incl. tables

Table 1: EPISKIN study data

Sample Tissue Replicate Optical Density (OD) OD - Blank % Negative Control
Negative Control a 0.885 0.753 100.7
0.845 0.712
b 0.869 0.737 96
0.793 0.661
c 0.907 0.775 103.4
0.862 0.73
Replicates a,b,c Average 0.728 100
sd 0.039 3.7
Positive Control a 0.434 0.302 40.3
0.418 0.285
b 0.233 0.1 12.5
0.214 0.082
c 0.288 0.156 20.2
0.27 0.138
Replicates a,b,c Average 0.177 24.3
sd 0.094 14.4
Methyl Cedryl Ketone a 0.708 0.576 77.6
0.685 0.553
b 0.672 0.539 71
0.627 0.494
c 0.728 0.596 79.9
0.7 0.568
Replicates a,b,c Average 0.554 76.2
sd 0.035 4.6
Blank 0.128
0.134
0.134
0.131
0.133
0.134
Average 0.132
sd 0.003

sd = Standard Deviation

Table 2:Summary of EPISKIN assay results

Sample Tissue viability as percentage of mean OD negative control
Replicate Tissues Mean ± Prediction
MTT endpoint
a b c SD
Negative Control 100.7 96 103.4 100.0 ± 3.7 Not applicable
Positive Control 40.3 12.5 20.2 24.3 ± 14.4 Irritant
Methyl Cedryl Ketone 77.6 71 79.9 76.2 ± 4.6 Non-irritant

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
It was concluded that the test substance, Methyl Cedryl Ketone, with a mean tissue viability of 76.2 ± 4.6%, was predicted as non-irritant to the skin.
Executive summary:

In an In Vitro Skin Irritation test Reconstructed Human Epidermis Test Method (MBB0004), reconstituted human epidermis skin cultured in vitro was exposed to 10 µL undiluted Methyl Cedryl Ketone for 15 ± 0.5 minutes.

Both the negative and positive control results met the assay acceptance criteria. The percentage mean viability of the Methyl Cedryl Ketone was 76.2 ± 4.6 of the negative control. As the mean tissue viability is greater than 50% of the negative control value, Methyl Cedryl Ketone is classed as non-irritant.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 439 in vitro skin irritation (Reconstructed Human Epidermis Test Method; EPISKIN™ Skin Irritation Test).

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