Reaction mass of (3aR,5aS,9aS,9bR)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan and (3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2012-10-02 to 2012-10-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Monitoring Programme (Inspected on 2012-07-10)

Test material

Test material information

Constituent 1

Reference substance name:

[3aR-(3aα,5aβ,9aα,9bβ)]-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan

EC Number:

229-861-2

EC Name:

[3aR-(3aα,5aβ,9aα,9bβ)]-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan

Cas Number:

6790-58-5

Molecular formula:

C16H28O

IUPAC Name:

(3aR,5aS,9aS,9bR)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Constituent 2

Reference substance name:

(3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Cas Number:

234431-64-2

Molecular formula:

C16H28O

IUPAC Name:

(3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Test material form:

solid: crystalline

Details on test material:

- Description: white crystalline solid
- Storage conditions: room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 12-EKIN-036
- Production date: not reported
- Shipping date: 02 October 2012
- Delivery date: 02 October 2012
- Date of initiation of testing: 02 October 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: not reported
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: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in DPBS
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm (without a reference filter)
- Filter: not applicable
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: no data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: Negative Control Item 0.630, 0.612, 0.691 (historical (124 studies) mean of the negative control was 0.797 ± 0.086; The lowest value was 0.603 and the highest was 1.315).
- Barrier function: IC50= 2.1 mg/mL ( ≥ 1.5 mg/mL)
- Morphology: well differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: : absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: the test item did not directly reduce MTT

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is less or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- Amount applied: 10±2 mg of the solid test item.
Formulations will be used within two hours of preparation and will be assumed to be stable for this period. The concentration, stability and homogeneity of the formulations will not be determined by analysis.

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 epidermis/product

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 h post-exposure incubation period

Value:

64.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6.8%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: not reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD540 for the negative control treated tissues was 0.644 and the standard deviation value of the percentage viability was 6.4%. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.8% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.2%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 7.6%. The test item acceptance criterion was therefore satisfied.
- Historical control data:
Since October 2009 to the commencement of this study (124 studies) the historical mean OD540 of the positive control was 0.059 ± 0.022 and the mean viability was 7.4 ± 3.2%. In this same period the historical mean OD540 of the negative control was 0.797 ± 0.086.
Also in the same historical period the lowest OD540 of the positive control was 0.025 and the highest 0.175 and the lowest percentage viability was 1.3 and the highest was 15.6. The lowest OD540 of the negative control was 0.603 and the highest was 1.315.
In this study the mean OD540 for the positive control was 0.044 and the mean viability was 6.8%. The mean OD540 of the negative control was 0.644.
The positive and negative controls were therefore within the historical ranges for this testing facility.

Any other information on results incl. tables

Table 7.3.1/1: Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD540 of tissues	Mean OD540 of triplicate tissues	±SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.630	0.644	0.041	97.8	100*	6.4
	0.612			95.0
	0.691			107.3
Positive Control Item	0.039	0.044	0.008	6.1	6.8	1.2
	0.053			8.2
	0.039			6.1
Test Item	0.372	0.416	0.049	57.8	64.7	7.6
	0.408			63.4
	0.469			72.8

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:

Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of ST 10 C 08 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 (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

This test was designed to be compatible with the Method B.46 of Commission Regulation (EC) No. 440/2008/EC and was performed in compliance with GLP.

Triplicate tissues were treated with ST 10 C 08 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 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‑labelled 96‑well plate. The optical density was measured at 540 nm.

The relative mean viability of the test item treated tissues was 64.7 ± 7.6 %, after the 15‑minute exposure period.

The quality criteria required for acceptance of results in the test were satisfied.

Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.