Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-18 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 439 without deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
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. certificate)
Remarks:
05 March 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): SAUGE SCLAREE ESS. / CLARY SAGE OIL
- Other name Salvia sclarea oil
- CAS No.: 8016-63-5
- EINECS-No.: 283-911-8
- Appearance: Pale yellow liquid
- Expiry date: Sep. 2018
- Date of Receipt: 18. Dec. 2015
- Batch no.: EE 86453
- Analytical purity: 100% wt UVCB substance
- Homogeneity: Homogeneous
- Storage condition of test material: Room temperature 20 ± 5 °C; test item was stored in a closed aluminium vessel at 15.2-20.9 °C away from light and humidity under inert gas.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Linalool consortium / EE 86453
- Physical state: Clear pale yellow liquid
- Date of receipt: 22 December 2015
- Expiration date of the lot/batch: December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature, protected from light and under nitrogen atmosphere

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: human reconstructed epidermis (tissues) reconstructed from normal human epidermal keratinocytes
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Species: Human reconstructed epidermis (tissues)
Supplier: Episkin, Lyon, France.
Selection: At receipt, the pH (colour of the agar medium) and temperature indicators were checked to ensure the good quality of the tissues before use.
Storage conditions: At receipt, the living Episkin™ tissues were kept at room temperature in their packaging until required.
Description: The Episkin™ model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra-structure and is functionally equivalent to human in vivo epidermis.

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 of the test item was applied to the epidermis surface
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL Dulbecco’s Phosphate-Buffered Saline (D-PBS)
- Concentration (if solution): Negative control was prepared by diluting D-PBS 10X to 1X in water for injections.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution
- Concentration (if solution): SDS was diluted in water for injection to 5% (w/v).
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes (± 1 minute).
Duration of post-treatment incubation (if applicable):
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 h.
Number of replicates:
One 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.

Test system

Type of coverage:
other: not applicable
Details on study design:
PRELIMINARY TESTS
Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.
Test for direct MTT reduction with the test item: 10 μL of the test item were added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution; a negative control was tested concurrently by adding 10 μL of water for injections to 2 mL of a 0.3 mg/mL freshly prepared MTT solution; both mixtures were incubated in darkness at 37 °C for 3 hours (± 5 minutes). Then the colour of the solutions obtained was evaluated.
Test for the detection of the colouring potential of the test item: The intrinsic colour or the ability of the test item to become coloured in contact with water (simulating a tissue humid environment) was evaluated by adding 10 μL of test item to 90 μL of water for injections in a transparent recipient. After 1 hour (± 3 minutes) of mixing, the presence and intensity of the coloration was evaluated.

MAIN TEST
Pre-incubation of the tissues on their day of arrival (Day 0): A volume of 2 mL of pre-warmed (at 37 °C) maintenance medium was added to 3 wells of a 12-well plates (one plate per item). Then, each Episkin™ tissue was transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at 37 °C, 5% CO2 in a humidified incubator for at least 24 hours.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The exposure of the tissues to the test and control items was performed at room temperature for 15 minutes (± 1 minute)
- Temperature of post-treatment incubation: Tissues were incubated at 37 °C, 5% CO2 in a humidified incubator for 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
- At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D-PBS to gently remove any residual test or control items. The rinsed tissues were transferred to wells containing 2 mL of maintenance medium in each well and the plates were incubated at 37 °C, 5% CO2 in a humidified incubator for 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of MTT solution (0.3 mg/mL)
- Incubation time: On Day 3, following the 42 h post-exposure incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h at 37 °C, 5 % CO2 in a humidified incubator. At the end of the MTT incubation period, tissues were incubated with 500 μL of acidic isopropanol. Then, to extract the formazan (reduced MTT) from the MTT-loaded tissues, each tube was stored after vortexing at +5°C and protected from light until Day 6 of the experiment.
- Optical density measurements (Day 6): At the end of the formazan extraction period, the optical density was measured at 570 nm using a plate reader.

NUMBER OF REPLICATE TISSUES: Triplicate tissues for test item, negative and positive controls

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
Classification of irritation potential is based upon relative mean tissue viability following the 15 - minute exposure period followed by the 42 - hour post-exposure incubation period
Relative mean tissue viability is ≤50%: Irritant
Relative mean tissue viability is >50%: Non-Irritant

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minute exposure period and 42 h post-exposure incubation period
Value:
70
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:
PRELIMINARY TESTS
Test for direct MTT reduction with the test item: The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on water-killed tissues in parallel to the main test.
Test for the detection of the colouring potential of the test item: During this test, as the water solution containing the test item did not change colour, the test item was found not to have a colouring potential. As a result, no additional controls were used in parallel to the main test.

MAIN TEST
- Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 70% with a standard deviation of 7% as assessed by the MTT assay. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response.
- Evaluation of the colouration of tissues at the end of the MTT incubation period: All test item-treated tissues appeared blue which was considered indicative for viable tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 7.3.1/1: Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

Group

Tissue No.

OD measurements

Mean

ODblank

cOD

Mean cOD

Viability (%)

 

1st

2nd

1st

2nd

Negative control

1

0.610

0.620

0.037

0.573

0.583

 0.578

89

2

0.740

0.759

0.703

0.722

0.712

110

3

0.689

0.693

0.652

0.656

0.654

101

Positive control

1

0.106

0.108

0.037

0.069

0.071

0.070

11

2

0.119

0.116

0.082

0.079

0.080

12

3

0.125

0.118

0.088

0.081

0.084

13

Test item

1

0.485

0.511

0.037

0.448

0.474

0.461

71

2

0.428

0.455

0.391

0.418

0.404

62

3

0.525

0.544

0.488

0.507

0.497

77

OD = optical density

cOD = blank corrected optical density

 

Table 7.3.1/2: Mean tissue viability and Standard Deviations for the test item, the negative and positive controls

 

Group

cOD

Viability (%)

Mean

SD

Mean

SD

Negative control

0.648

0.067

100

10

Positive control

0.078

0.007

12

1

Test item

0.454

0.047

70

7

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item is considered to be non-irritant to skin. According to the results of this study, the classification of the test item should be: not classified (Directive 67/548/EEC) and no category (Regulation (EC) No. 1272/2008).
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.

 

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Test item, SAUGE SCLAREE ESS. / CLARY SAGE OIL was applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 h at 37 °C, 5 % CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100 % (reference viability).

 

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.

 

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

 

All test item-treated tissues appeared blue which was considered indicative for viable tissues. Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 70% with a standard deviation of 7% as assessed by the MTT assay. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response.

 

Under the experimental conditions of this study, the test item is considered to be non-irritant to skin. According to the results of this study, the classification of the test item should be: not classified (Directive 67/548/EEC) and no category (Regulation (EC) No. 1272/2008).