1,3,4,6,7,8a-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalen-8(5H)-one

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 09 April 2014 and 14 April 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2013

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:

1,3,4,6,7,8a-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalen-8(5H)-one

EC Number:

245-890-3

EC Name:

1,3,4,6,7,8a-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalen-8(5H)-one

Cas Number:

23787-90-8

Molecular formula:

C15H24O

IUPAC Name:

1,1,5,5-tetramethylhexahydro-2H-2,4a-methanonaphthalen-8(5H)-one

Test material form:

other: Liquid

Details on test material:

Identification: Isolongifolanone
Batch: SC00010626
CAS number: 23787-90-8
EINECS: 245-890-3
Chemical name: 2,2,7,7-TETRAMETHYLTRICYCLO[6.2.1.0^{1,6}]UNDECAN-5-ONE
Purity: 69.9%
Physical state/Appearance: extremely pale yellow liquid
Expiry Date: 17 Febraury 2016
Storage Conditions: room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Reconstructed human epidermis model

Control samples:

other: Yes

Test animals

Species:

other: EPISKIN™ Reconstructed Human Epidermis Model

Strain:

other: Not applicable

Test system

Type of coverage:

other: Not applicable

Preparation of test site:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Not applicable

Duration of treatment / exposure:

15 minutes

Observation period:

42 Hour post-exposure incubation period

Number of animals:

Not applicable

Details on study design:

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.

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, each 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, 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.


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 centre). After 7 Minutes 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. The plate(s) 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 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 plate(s). 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.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other:

Value:

33.3

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes exposure. Remarks: Classified as irritant. (migrated information)

In vivo

Irritant / corrosive response data:

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.


Test Item, Positive Control Item and Negative Control Item
The relative mean viability of the test item treated tissues was 33.3% after a 15 Minute exposure period and 42 hours post exposure incubation period.

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

Any other information on results incl. tables

Mean OD₅₆₂Values and Percentage 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	1.316	1.247	0.061	105.5	100*	4.9
	1.228			98.5
	1.198			96.1
Positive Control Item	0.047	0.051	0.003	3.8	4.1	0.3
	0.053			4.3
	0.052			4.2
Test Item	0.532	0.414	0.170	42.7	33.3	13.7
	0.492			39.5
	0.219			17.6

SD= Standard deviation

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

OD₅₆₂= Optical Density

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 4.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.3%. The positive control acceptance criterion was therefore satisfied.

 

The mean OD₅₆₂for the negative control treated tissues was 1.247 and the standard deviation value of the percentage viability was 4.9%. The negative control acceptance criterion was therefore satisfied.

 

The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 13.7%. The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item 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 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. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42‑Hour post‑exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end‑point can be used to either confirm a non-irritant result or will be used to override the non‑irritant result.

 

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 was33.3% 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.

  

Conclusion

The test item was classified as irritant. The following classification criteria apply:

EU DSD (67/548/EEC) Irritant requires symbol “Xi” risk phrase R38 “Irritating to Skin”.
EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2.