Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 18 May 2016 and 23 May 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 439 in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 28 July 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

EC Number:

944-405-9

Molecular formula:

C11H20O2

IUPAC Name:

Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

Test material form:

liquid

Details on test material:

- Substance name as cited in test report: FRET 13-0156
- Phystical state: clear, yellowish liquid
- Storage conditions: ambient temperature (15-25 °C), protected from light

Specific details on test material used for the study:

Identification: FRET 13-0156

SOURCE OF TEST MATERIAL
- Appearance/physical state: Amber colored liquid
- Source and lot/batch No. of test material: PDJ378-33
- Expiration date of the lot/batch: 01 March 2018
- Purity: 95.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France.

Source strain:

other: not applicable.

Details on animal used as source of test system:

Not applicable.

Justification for test system used:

Not applicable.

Vehicle:

unchanged (no vehicle)

Details on test system:

Preparation of Negative and Positive Control Items and MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)
The negative control item, Dulbecco’s Phosphate Buffered Saline (DPBS) with Mg2+ and Ca2+ ions, was used as supplied.
The positive control item, sodium dodecyl sulphate (SDS), was prepared as a 5% (w/v) aqueous solution.
A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

Test System (EPISKIN™ Reconstructed Human Epidermis Model Kit)
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 18 May 2016
EpiSkinTM Tissues (0.38 cm2) lot number: 16-EKIN-020
Maintenance Medium lot number: 16-MAIN3-034
Assay Medium lot number: 16-ESSC-020

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay: The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction: As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
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 or purple, 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.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

Pre-incubation (Day 1: 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 for two hours.

Main Test

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. 10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% (w/v) served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes. 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 Ca2+ and Mg2+. 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 Loading/Formazan Extraction (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. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The 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.

Data Evaluation

Quantitative MTT Assessment (Percentage Tissue Viability)
For the test item the relative mean tissue viabilities obtained after the 15 Minute exposure period followed by the 42 Hour post exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562 of test item/mean OD562 of negative control) x 100
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 is shown under 'Any other information on materials and methods incl. tables'.

Quality Criteria
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
Test Item:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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. 10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface.

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period each tissue was rinsed before incubating for 42 hours.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

4.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The relative mean viability of the test item treated tissues was 4.2% after a 15 Minute exposure period and 42 Hour post exposure incubation period.

Other effects / acceptance of results:

Refer to ‘Any other information on results incl. tables’.

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

 

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

 

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD₅₆₂values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in the table below. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in the table below

The relative mean viability of the test item treated tissues was 4.2% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.

It was considered unnecessary to perform IL-1aanalysis as the results of the MTT test were unequivocal.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 4.2% relative to the negative control treated tissues and the standard deviation value of the viability was 1.0%.The positive control acceptance criteria were therefore satisfied.

The mean OD₅₆₂for the negative control treated tissues was 1.177 and the standard deviation value of the viability was 2.2%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 0.7%. The test item acceptance criterion was therefore satisfied.

Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562of tissues	Mean OD562of triplicate tissues	±SDof OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	1.204	1.177	0.026	102.3	100*	2.2
	1.152			97.9
	1.174			99.7
Positive Control Item	0.051	0.049	0.012	4.3	4.2	1.0
	0.037			3.1
	0.060			5.1
Test Item	0.048	0.049	0.008	4.1	4.1	0.7
	0.041			3.5
	0.057			4.8

OD = Optical Densit

SD = Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item FRET 13-0156 was classified as irritant. The following classification criteria apply: EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2.

Executive summary:

Introduction

The purpose of this test, conducted according to OECD Test Guideline 439, was to evaluate the skin irritation potential of the test item using the EPISKIN^TMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

 

Method

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).

 

Results

The relative mean viability of the test item treated tissues was 4.1% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.