[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 December 2014 to 09 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 439 with minor deviations: historical data and tissues certificate are not reported

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

historical data and tissues certificate are not reported

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

historical data and tissues certificate are not reported

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on 12-14 March 2014 / Signed on 12 May 2014)

Test material

Test material information

Constituent 1

Reference substance name:

(+)-(2Z)-4,8-DIMETHYL-2,7-NONADIEN-4-OL

Molecular formula:

C11H120

IUPAC Name:

(+)-(2Z)-4,8-DIMETHYL-2,7-NONADIEN-4-OL

Constituent 2

Reference substance name:

(-)-(2Z)-4,8-DIMETHYL-2,7-NONADIEN-4-OL

Molecular formula:

C11H20O

IUPAC Name:

(-)-(2Z)-4,8-DIMETHYL-2,7-NONADIEN-4-OL

Test material form:

liquid

Details on test material:

- Physical state: Colorless liquid
- Storage condition of test material: Stored at 2-8 °C temperature and protected by Nitrogen.

Specific details on test material used for the study:

- Purity test date: 04 November 2014
- Storage condition of test material: Refrigerated at 4°C in the dark under nitrogen

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: no data

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 03 December 2014
EpiSkinTM Tissues (0.38cm2) lot number: 14-EKIN-046
Maintenance Medium lot number: 14-MAIN3-052
Assay Medium lot number: 14-ESSC-049

TEST FOR DIRECT MTT REDUCTION
- 10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
- If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

PRE-INCUBATION (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

APPLICATION OF TEST ITEM AND RINSING (DAY 1)
- 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering.

REMOVAL OF TEST MATERIAL AND CONTROLS
- At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. 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.
- The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE (DAY 3)
- Following the 42 - hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
- MTT concentration: Tissues were transferred to the MTT filled wells (0.3 mg/mL MTT solution)
- Incubation time: Tissues were incubated for 3 hours at 37 °C, 5% CO2 in air
- At the end of the 3 - hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 6)
- At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
- For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

PREDICTION MODEL / DECISION CRITERIA
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

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 (26.3 μL /cm2)of the test item was applied to the epidermis surface.
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL of DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL of SDS 5% w/v

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes at room temperature.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 hours.

Duration of post-treatment incubation (if applicable):

- On Day 3, at the end of the 42 h post-treatment incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h at 37 °C, 5 % CO2 in air.
- On Day 6, at the end of the formazan extraction period: The optical density was measured at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Number of replicates:

Triplicate tissues for test item, negative and positive controls

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:

9.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

TEST ITEM
The relative mean viability of the test item treated tissues was 9.9% after a 15 - minute exposure period and 42 hours post-exposure incubation period.
The mean concentration of inflammatory mediator IL-1α in the culture medium retained from the test item treated tissues was 164.375 pg/mL.

OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 0.868 and the standard deviation value of the percentage viability was 1.3%. 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 10.7% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.4%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 1.8%. The test item acceptance criterion was therefore satisfied.

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	0.871	0.868	0.011	100.3	100*	1.3
	0.878			101.2
	0.856			98.6
Positive Control Item	0.082	0.093	0.012	9.4	10.7	1.4
	0.106			12.2
	0.090			10.4
Test Item	0.080	0.086	0.015	9.2	9.9	1.8
	0.074			8.5
	0.103			11.9

 

SD = Standard deviation

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

OD562 = Optical Density

 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test item is classified as skin irritant (Category 2) according to Regulation (EC) No 1272/2008 (CLP) and to GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN ™ 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 9.9% after the 15-Minute exposure period and 42 hours post-exposure incubation period.

The mean concentration of inflammatory mediator IL-1α in the culture medium retained from the test item treated tissues was 164.375 pg/mL.

 

The quality criteria required for acceptance of results in the test were satisfied.

 

Under the experimental conditions of this study, the test item is classified as skin irritant (Category 2) according to Regulation (EC) No 1272/2008 (CLP) and to GHS.