ethyl 2-[[(1R,2S,5R)-5-methyl-2-propan-2-ylcyclohexanecarbonyl]amino]acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-{[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]formamido}acetate

EC Number:

695-735-2

Cas Number:

68489-14-5

Molecular formula:

C15H27NO3

IUPAC Name:

ethyl 2-{[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]formamido}acetate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Symrise, WS-5, 83100003
- Expiration date of the lot/batch: 09 December 2018
- Purity test date: 99.76%
- Physical state/Appearance: White solid

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:

other: three-dimensional reconstructed human epidermis model (EPISKINTM model)

Cell source:

other: adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.

Details on animal used as source of test system:

not applicable

Justification for test system used:

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.
The procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study and acceptable to the current OECD guideline.

Vehicle:

unchanged (no vehicle)

Remarks:

neat test item WS-5

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM reconstructed human epidermis model
- Supplier: SkinEthic Laboratories, Lyon, France
- EpiSkinTM Tissues (0.38cm2) lot number: 17-EKIN-028
- Date received: 11 July 2017
Maintenance Medium lot number: 17-MAIN3-028
Assay Medium lot number :17-ESSC-027

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): at 37 C, 5% CO2 in air

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 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.
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: Labtech LT-4500 microplate reader, (LT-4500 IFU DOC Revision No.1.0)
- Wavelength: 570 nm (without a reference filter).

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
To identify the possible interference with the MTT endpoint, WS-5 was checked for the ability to directly reduce MTT according to the following procedure:
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 mg 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.

PREDICTION MODEL / DECISION CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria (EpiskinTM SOP February 2009 Version 108 ECVAM Skin Irritation validation Study) 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 OD570 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:

TEST MATERIAL
- Amount(s) applied: Approximately 10 mg (26.3 mg/cm2) of WS-5.
The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis.

NEGATIVE CONTROL
- Concentration (if solution): 10 µL of DPBS.

POSITIVE CONTROL
- Concentration (if solution): 10 µL of SDS 5% w/v
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.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours at 37C, 5% CO2 in air

Number of replicates:

3

Test system

Details on study design:

TEST SITE
- Area of exposure: Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface.
The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: at the end of the exposure period (after 15 min)


SCORING SYSTEM:
- Method of calculation:

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 OD570of test item / OD570of negative control) X 100

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

ca. 63.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

22.7%

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

other: Mean OD570

Remarks:

triplicate tissues

Run / experiment:

2

Value:

ca. 0.396

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.621

Positive controls validity:

valid

Remarks:

0.141

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Table 1. 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:

Criteria for in vitro interpretation	Prediction	EU CLP (Regulation (EC) No 1272/2008)	UN GHS
Relative mean tissue viability is ≤50%	Irritant	H315 Category 2	H315 Category 2
Relative mean tissue viability is >50%	Non-irritant	Not classified for irritation	Not classified or UN GHS Category 3 can not be determined

Table 2. 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 DPBS	0.581	0.621	0.051	93.6	100*	8.2
	0.604			97.3
	0.679			109.3
Positive Control Item SDS	0.212	0.141	0.069	34.1	22.7	11.0
	0.137			22.1
	0.075			12.1
Test Item WS-5	0.334	0.396	0.070	53.8	63.8	11.2
	0.382			61.5
	0.471			75.8

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:

other: not classified for Irritation according to EU CLP

Conclusions:

Based on available data on skin irritaiton performed according the OECD TG 439, WS-5 is not classified for skin irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Executive summary:

The purpose of this study performed according to OECD Test Guideline 439 was to evaluate the skin irritation potential of the test item, WS-5 using the EPISKIN TM reconstructed human epidermis model.

In the direct MTT Reduction assay, WS-5 did not turn blue or purple which indicated that the test item did not directly reduce MTT. The solution containing WS-5 produced no colour. It was therefore unnecessary to run colour correction tissues.

Triplicate human skin tissues were treated with the neat test item, WS-5 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. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing 500 µL 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.

The relative mean viability of WS-5 treated tissues was 63.8% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period. It was therefore considered unnecessary to perform IL-1 alpha analysis as the results of the MTT test were unequivocal.

Conclusions:

Based on the above results, the test item, WS-5 was classified as non-irritant according to OECD Test Guideline 439. 

The following classification criteria apply:

EU DSD and CLP - not classified for Irritation.

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