[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:

Experimental start date 19 August 2020 Experimental completion date 24 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2(R/S)‐1‐{[(1R,2S,5R,8S)‐4,4,8‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐1‐yl]oxy}pentan‐2‐o and 2(R/S)‐1‐{[(1S,2S,5R,8R)‐1,4,4‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐8‐yl]oxy}pentan‐2‐ol

Molecular formula:

C20H36O2

IUPAC Name:

Reaction mass of 2(R/S)‐1‐{[(1R,2S,5R,8S)‐4,4,8‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐1‐yl]oxy}pentan‐2‐o and 2(R/S)‐1‐{[(1S,2S,5R,8R)‐1,4,4‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐8‐yl]oxy}pentan‐2‐ol

Test material form:

liquid: viscous

Details on test material:

Chemical name: 1‐[(4,4,8‐trimethyltricyclo[6.3.1.02,5]dodecan‐1‐yl)oxy]pentan‐2‐ol and 1‐[(1,4,4‐trimethyltricyclo[6.3.1.02,5]dodecan‐8‐yl)oxy]pentan‐2‐ol and isomers

Specific details on test material used for the study:

Identification: FRET 18-0091
Chemical Name: 1-[(4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1-yl)oxy]pentan-2-ol and 1-[(1,4,4-trimethyltricyclo[6.3.1.02,5]dodecan-8-yl)oxy]pentan-2-ol and isomers
Batch: RDEA571-42
Purity: 92.0%
Physical state/Appearance: Light yellow viscous liquid
Expiry Date: 31 December 2021
Storage Conditions: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

other: EPISKINTM reconstructed human epidermis model

Cell type:

other: EPISKINTM reconstructed human epidermis model

Cell source:

other: EPISKINTM reconstructed human epidermis model

Source strain:

other: EPISKINTM reconstructed human epidermis model

Details on animal used as source of test system:

Supplier : EpiSkin Laboratories, Lyon, France
Date received : 18 August 2020
EpiSkinTM Tissues (0.38cm2) lot number : 20-EKIN-034
Maintenance Medium lot number : 20-MAIN3-023
Assay Medium lot number : 20-ESSC-023

Justification for test system used:

The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Following a full validation study (ECVAM, 2009) the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

Vehicle:

unchanged (no vehicle)

Details on test system:

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. 10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface ensuring uniform covering. 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 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 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 ( 35 to 10 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 2 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 (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT 4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

130.1

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:

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

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 130.1% after a 15 Minute exposure period and 42 Hour post exposure incubation period.

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

Acceptance Criteria
The relative mean tissue viability for the positive control treated tissues was 7.5% relative to the negative control treated tissues and the standard deviation value of the viability was 0.3%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.665 and the standard deviation value of the viability was 4.6%. 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 24.4%. The test item acceptance criterion was therefore not satisfied. The standard deviation was considered acceptable as each of the three identically treated replicates were clearly negative compared to the negative control. This is reported as a deviation.

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

Any other information on results incl. tables

Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570of tissues	Mean OD570of triplicate tissues	±SDof OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.665	0.665	0.031	100.0	100*	4.6
	0.696			104.7
	0.635			95.5
Positive Control Item	0.049	0.050	0.002	7.4	7.5	0.3
	0.052			7.8
	0.048			7.2
Test Item	1.049	0.865	0.163	157.7	130.1	24.4
	0.805			121.1
	0.741			111.4

 

OD= Optical Density

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:

In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

Introduction

The purpose of this test 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 is 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 was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple 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 570 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 130.1% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied with the exception that the standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 24.4% which was higher than the 18% cut-off. However, the standard deviation was considered acceptable as each of the three identically treated replicates were clearly negative compared to the negative control. This is reported as a deviation.

 

Conclusion

In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).