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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 18 to 24, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 439 and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)

Test material

Constituent 1
Chemical structure
Reference substance name:
(3R)-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]-3-hexanol
Cas Number:
253454-12-5
Molecular formula:
C15H30O
IUPAC Name:
(3R)-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]-3-hexanol
Constituent 2
Chemical structure
Reference substance name:
(3S)-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]-3-hexanol
Cas Number:
253454-10-3
Molecular formula:
C15H30O
IUPAC Name:
(3S)-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]-3-hexanol
Test material form:
liquid
Details on test material:
- Physical state: Colourless liquid
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature, protected from light, in the original container (aluminium bottle)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 14-EKIN-005
- Production date: 18 February 2014
- Shipping date: 18 February 2014
- Delivery date: 18 February 2014
- Date of initiation of testing: 18-24 February 2014
- Expiry date: 24 February 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ambient temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Not reported. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/L
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: without reference filter
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function: 2.2 mg/mL ( ≥ 1.5 mg/mL)
- Morphology: well differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination:
Absence of HIV1 and 2 antobodies, hepatitis C antibodies, hepatitis B antigen HBs
Absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

NUMBER OF REPLICATE TISSUES: triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : the test item did not directly reduce MTT

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is less or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
15 minutes at room temperature
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Triplicate

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
63.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: the test item was not able to directly reduce MTT
- Colour interference with MTT: no colour interference were observed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.013 and the standard deviation value of the percentage viability was 2.4%. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 8.4%. The test item acceptance criterion was therefore satisfied.
For the previous 35 experiments conducted between May 2013 and February 2014 using this test method, the mean OD of the positive control was 0.084 ± 0.036 and the mean percentage viability was 9.6 ± 4.4. In this same period the mean OD of the negative control was 0.898 ±0.106.
The positive control reflected the ability to respond to an irritant test item under the conditions of the test. The negative control in this study was greater than the mean OD of the negative control in the previous 35 experiments conducted with this test method.

Any other information on results incl. tables

Table 7.3.1/1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

1.034

1.013

0.024

102.1

100*

2.4

1.018

100.5

0.986

97.3

Positive Control Item

0.062

0.054

0.011

6.1

5.3

1.1

0.042

4.1

0.058

5.7

Test Item

0.687

0.646

0.085

67.8

63.7

8.4

0.702

69.3

0.548

54.1

SD=        Standard deviation

*=         The mean viability of the negative control tissues is set at 100%

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 63.7% after the 15‑minute exposure period and 42 hours post‑exposure incubation period.

 

The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1%. The positive control acceptance criterion was therefore satisfied. The mean OD562 for the negative control treated tissues was 1.013 and the standard deviation value of the percentage viability was 2.4%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 8.4%. The test item acceptance criterion was therefore satisfied.

 

Under the test conditions, test item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.