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

Endpoint summary

Description of key information

In the key study performed according to OECD Test Guideline 439, the skin irritation potential of WS-5 was evaluated using the EPISKIN TM reconstructed human epidermis model.

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, toptical 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.

Based on these results, WS-5 was classified as non-irritant according to OECD Test Guideline 439. 

In the key in vitro Bovine Corneal Opacity and Permeability (BCOP) study performed according to OECD TG 437, WS-5 was applied to assess

any eye damage and e changes in corneal opacity and permeability . All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Based on the In Vitro irritancy score (IVIS=0.0), no category can be applied for WS-5. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

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

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

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

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

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%

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: WS-5, 83100003
- Expiration date of the lot/batch: 12 September 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

Species:

other: Eyes from adult cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source of Bovine Eyes
- Source: adult cattle
- Age: typically 12 to 60 months old

Conditions:
The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: Approximately 0.53 g of the solid test item, WS-5

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

The condition of the cornea was visually assessed post- treatment

Number of animals or in vitro replicates:

3 in vitro replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS : All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea.
The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.

QUALITY CHECK OF THE ISOLATED CORNEAS : The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES : 3 replicates

NEGATIVE CONTROL USED : Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Sodium chloride 0.9% w/v was used for negative control purposes.

SOLVENT CONTROL USED (if applicable) - no

POSITIVE CONTROL USED : Three corneas were allocated to the positive control item. 20% w/v Imidazole was used for positive control purposes.

APPLICATION DOSE AND EXPOSURE TIME : Approximately 0.53 g of the solid test item, WS-5 was found to adequately cover the corneal surface. 0.75 ml of each negative and positive control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated uppermost, at 32 ± 1 ºC for 240 minutes.

TREATMENT METHOD: anterior chamber

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) . The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

DECISION CRITERIA:
For an acceptable test the following positive control criterion should be achieved:
20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2015 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 50.8 to 100.4.

For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2015 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤5.4 and for permeability ≤0.070.

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

ca. 0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤5.4 and permeability ≤0.070. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore satisfied.

Table 1. The results of the In Vitro irritancy scores (IVI):

Treatment	In Vitro Irritancy Score
Test Item	0.0
Negative Control	2.4
Positive Control	73.9

Interpretation of results:

other: Not requiring classification according to EU CLP

Conclusions:

Based on available data on eye irritaiton performed according the OECD TG 437, WS-5 is not classified for eye irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Executive summary:

The purpose of The Bovine Corneal Opacity and Permeability (BCOP) testing performed according to OECD TG 437, was to identify, whether WS-5 can induce a serious eye damage. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

The corneas treated with the test item were clear post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate anIn Vitro Irritancy Score. Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

The positive control In Vitro Irritancy Score was within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤5.4 and permeability ≤0.070. The negative control acceptance criteria were therefore satisfied.

Based on the In Vitro irritancy score (IVIS), no category is applied for WS-5. Therefore, WS-5 is not classified as eye irritant according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on available data on skin and eye irritaiton performed according the OECD TG 439 and OECD TG 437, WS-5 is not classified for skin or eye irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.