Decahydro-2-naphthyl 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:

15 February 2016 - 22 February 2016

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)

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation 440/2008, 1st ATP 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Decahydro-2-naphthyl acetate

EC Number:

234-054-3

EC Name:

Decahydro-2-naphthyl acetate

Cas Number:

10519-11-6

Molecular formula:

C12H20O2

IUPAC Name:

decahydronaphthalen-2-yl acetate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Epidermal keratinocytes

Source strain:

other: Human

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Human
- Tissue: normal epidermal keratinocytes

Justification for test system used:

Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. Skin irritation is defined in Section 3.2.1.1 of Annex I to the CLP regulation as “[…] the production of reversible damage of the skin following the application of a test substance for up to 4 hours”

According to Annex VII of the REACH Regulation, if new test data are required these must be derived from in vitro methods only. The EpiDerm™ human skin model (OECD 439) is an accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis
- Source: MatTek Corporation (82105 Bratislava, Slovakia)
- Tissue batch number(s): 23314
- Date of initiation of testing: 15 February 2016
- Pre-warming: Prior to exposure the EpiDerm™ tissues were inspected for quality.

EPIDERM™ QUALITY CRITERIA
- Air bubbles between agarose and insert were not >30% of the total surface
- Liquid on top of the insert was removed with sterile cotton tips
- If moisture was observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: DPBS, at least 15 times
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: An older version (11/07/14) of the performing laboratory protocol was valid instead of a newer one (26/03/2016)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 24 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 ± 1 nm
- Filter: 570 ± 1 nm filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Manufacturer MTT assay, 1.683 ± 0.077 OD (acceptable range: 1.0 to 3.0)
- Barrier function: Manufacturer ET-50 assay, 6.55 hrs (acceptable range: 4.77 to 8.72 hrs)
- Contamination: Manufacturer long term antibiotic and antimycotic free culture, sterile (acceptable criteria: no contamination)

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: The test chemical is considered to be irritant to skin accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is ≤ 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 (corresponding to 47 μL/cm2)
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% SLS solution in deionised water

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

43.5 hours

Number of replicates:

3 per test item treatment, positive control and negative control

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: relative absorbance

Run / experiment:

1

Value:

85

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

other: relative absorbance

Run / experiment:

2

Value:

107.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

other: relative absorbance

Run / experiment:

3

Value:

107.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean OD was 1.563 (criteria: ≥ 0.8 and ≤ 2.8 for 60 minutes treatment)
- Acceptance criteria met for positive control: Derease in the relative absorbance as compared to the negative control was 5.1% (criteria: ≤ 20%)
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability of the test item, the positive and negative controls in the main test ≤13% (criteria: < 18%)
- Range of historical values if different from the ones specified in the test guideline: 4.00 to 5.90% mean viability for positive control

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative absorbance value of the test item, corresponding to the cell viability did not decrease (100.0%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Executive summary:

Skin irritation of the test item was evaluated with the EpiDerm Reconstructed Human Epidermis Model. Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt.

Undiluted test item was applied to the EpiDerm tissue for 60 minutes, alongside a negative and positive control. The mean relative absorbance value of the test item, corresponding to the cell viability did not decrease (100.0%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD Test Guideline 439 and GLP, the study is considered to be reliable without restriction (Klimisch 1).