[(3aS*,4R*,5S*,7R*,7aS*)-4,7-methano-2,3,3a,4,5,6,7,7a-octahydro-1H-inden-5-yl]methyl acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UN GHS

Version / remarks:

3rd revision

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[(3aS*,4R*,5S*,7R*,7aS*)-4,7-methano-2,3,3a,4,5,6,7,7a-octahydro-1H-inden-5-yl]methyl acetate

Molecular formula:

C13H20O2

IUPAC Name:

[(3aS*,4R*,5S*,7R*,7aS*)-4,7-methano-2,3,3a,4,5,6,7,7a-octahydro-1H-inden-5-yl]methyl acetate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT Kit
- Tissue batch number(s): 23314
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 16 February 2016
- Date of initiation of testing: Pre-incubation phase started 16 February 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 ºC
- Temperature of post-treatment incubation (if applicable): 37 ± 1 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 300 μL, one washing step
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Not applicable, microplate reader
- Wavelength: 570 ± 1 nm
- Filter: Not reported
- Filter bandwidth: 570 ± 1 nm
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive control; 4.77 %
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: None reported
- Reproducibility: Relative standard deviation for mean viability; 14.79 % postive control. Relative standard deviation for mean absorption; 16.6 % postive control, 8.55 % negative control.

NUMBER OF REPLICATE TISSUES: 3 per control and test item treatment

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues Not applicable
- Procedure used to prepare the killed tissues (if applicable): Not applicable
- N. of replicates : Not applicable
- Method of calculation used: Not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 43.5 hours exposure is less than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- Concentration (if solution): Undiluted test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): Not reported

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5 % SLS in deioinsied water; concentration not reported

Duration of treatment / exposure:

43.5 hours

Duration of post-treatment incubation (if applicable):

67.5 hours

Number of replicates:

3 per test item, negative control or postive control

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: Mean absorbance of three wells blank corrected

Run / experiment:

Tissue 1

Value:

0.601

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

other: Mean absorbance of three wells blank corrected

Run / experiment:

Tissue 2

Value:

0.592

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

other: Mean absorbance of three wells blank corrected

Run / experiment:

Tissue 3

Value:

0.585

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

other: Mean absorbance of three wells blank corrected

Run / experiment:

Mean

Value:

0.593

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation / corrosion parameter:

other: Relative absorbance (%) compared to blank

Run / experiment:

Tissue 1

Value:

38.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation / corrosion parameter:

other: Relative absorbance (%) compared to blank

Run / experiment:

Tissue 2

Value:

37.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation / corrosion parameter:

other: Relative absorbance (%) compared to blank

Run / experiment:

Tissue 3

Value:

37.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 8

Irritation / corrosion parameter:

other: Relative absorbance (%) compared to blank

Run / experiment:

Mean

Value:

37.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Standard deviation: 1.4 %

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: Did not show blue colour after 1 hour incubation
- Colour interference with MTT: No colour change

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, within the required range of OD ≥ 0.8 and ≤ 2.8
- Acceptance criteria met for positive control: Yes, induced a decrease in absorbance of 5.1 % compared to the negative control

Any other information on results incl. tables

Results of treatment with test item and the controls

Dose Group	Exposure Interval	Tissue No.	Absorbance 570 nm (Well 1)	Absorbance 570 nm (Well 2)	Absorbance 570 nm (Well 3)	Mean Absorbance of 3 Wells*	Mean Absorbance of three wells blank corrected	Mean Absorbance of three tissues after blank correction	Rel. Absorbance (%) 1, 2 + 3**	Relative Standard Deviation (%)	Mean. Rel. Absorbance (% of negative control)***
Blank			0.037	0.038	0.038	0.038	0.000
Negative Control	60 mins	1	1.540	1.548	1.508	1.532	1.495	1.563	95.6	4.1	100.0
		2	1.644	1.591	1.593	1.609	1.572		100.6
		3	1.717	1.631	1.630	1.659	1.622		103.8
Positive Control	60 mins	1	0.117	0.120	0.118	0.118	0.081	0.080	5.2	4.4	5.1
		2	0.118	0.107	0.116	0.113	0.076		4.8
		3	0.104	0.132	0.124	0.120	0.083		5.3
Test item	60 mins	1	0.646	0.628	0.643	0.639	0.601	0.593	38.5	1.4	37.9
		2	0.617	0.645	0.627	0.629	0.592		37.9
		3	0.613	0.625	0.630	0.623	0.585		37.4

 * Mean of three replicate wells after blank correction

** relative absorbance per tissue (rounded values): 100*(absorbance_tissue)/ (mean absorbance_{negative control})

***relative absorbance per treatment group (rounded values): 100*(mean absorbance_{test item/positive control})/ (mean absorbance_{negative control})

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

and EU CLP criteria

Conclusions:

The test item is a category 2 irritant to skin according to UN GHS regulations based on a human skin model test conducted following OECD 439, EU B.46 and UN GHS guidelines and performed according to GLP.

Executive summary:

The test item was assessed for skin irritation in a human skin model test conducted following OECD 439, EU B.46 and UN GHS guidelines in undiluted form alongside a postive and negative control. The results of the test indicate that the test item is a Category 2 irritant to skin according to the UN GHS regulations.

The study is a GLP compliant guideline experiment study available as an unpublished study report. The study has no restrictions and is fully adequate for assessment.