Reaction mass of 2,4-dimethyl-3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-ol and 3,4-dimethyl-3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-ol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 23, 2016 - January 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Reaction mass of 2,4-dimethyl-3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-ol and 3,4-dimethyl-3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-ol

EC Number:

944-067-2

Molecular formula:

C12H18O

IUPAC Name:

Reaction mass of 2,4-dimethyl-3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-ol and 3,4-dimethyl-3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-ol

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Normal Human-Derived Epidermal Keratinocytes

Cell source:

other: SkinEthic Laboratories (69007 Lyon, France)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin
- Tissue batch number(s): 17-EKIN-002
- Delivery date: January 10, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: not specified
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsed with PBS until all test material was removed
- 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: 3h
- Spectrophotometer: Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Historical neg control OD= 0.61-1.52, Current negative control OD = 1.053

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure is greater 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): 10 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% SLS solution in deionised water

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: Mean relative tissue viability (%)

Value:

6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: none
- Colour interference: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, after treatment with the negative control the mean absorbance value of the triplicate tissues was 1.053 which was well within the required acceptability criterion of mean OD >= 0.6 till ≤ 1.5 thus showing the quality of the tissues.
- Acceptance criteria met for positive control: yes, treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 8.1% (<40%) thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: yes, the rel. standard deviations between tissues of the same treatment group were below 16% (<18%), thus ensuring the validity of the study.
- Range of historical values if different from the ones specified in the test guideline:

Data of 232 studies performed from October 2007 until May 2014

Positive Control
Mean Viability: 19.30%
Rel. Standard Deviation: 10.00%
Range of Viabilities: 1.70% - 35.40%

Negative Control
Mean OD: 1.01
Rel. Standard Deviation: 0.22
Range of ODs: 0.61 – 1.52

Data of 12 studies performed from July 2015 to January 2017

Positive Control
Mean Viability: 15.83%
Rel. Standard Deviation: 11.88%
Range of Viabilities: 4.25% - 32.7%

Negative Control
Mean OD: 0.80
Rel. Standard Deviation: 0.22
Range of ODs: 0.61 – 1.09

Any other information on results incl. tables

The test for colour interference and direct MTT reduction was found to be negative, as 1) the colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item and 2) the colour of a test solution containing test substance and MTT did not turn blue/purple after incubation.

Applicant's summary and conclusion

Interpretation of results:

other: Skin Irritant Category 2

Remarks:

based on EU CLP (EC 1272/2008 and its updates)

Conclusions:

Under the conditions of this test, the relative mean tissue viability for the test item was determined to be 6%. This value is below the threshold for irritancy of ≤50%. Therefore, the test substance is considered to be a skin irritant.

Executive summary:

This in vitro skin irritation study to assess the irritation potential of Dimeth Cyclormol by means of the Human Skin Model Test, was performed in line with OECD439 and GLP principles. The test item did not directly reduce MTT, and did not cause colour interference. Triplicate tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. After washing, and further incubation for approximately 42 hours, tissue viability was determined by measuring MTT reduction. The viability and OD values of respectively the positive and negative control tissues were considered valid. After treatment with the test item the mean relative viability decreased to 6.0%. The value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered a skin irritant.