[(8R,9S,10R,13S,14S,17R)-3-ethoxy-17-ethynyl-13-methyl-2,7,8,9,10,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthren-17-yl] acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2015 - March 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(17alpha)-3-ethoxy-19-norpregna-3,5-dien-20-yn-17-ol

EC Number:

619-217-2

Cas Number:

96487-85-3

Molecular formula:

C22H30O2

IUPAC Name:

(17alpha)-3-ethoxy-19-norpregna-3,5-dien-20-yn-17-ol

Test material form:

solid: particulate/powder

Details on test material:

Appearance: off-white powder
Test item storage: at room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on animal used as source of test system:

Test system
EpiDerm Skin Model (EPI-200, Lot no.: 23271 kit EE and FF).
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Source
MatTek Corporation, Ashland MA, U.S.A.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200, kit EE and FF).
- Tissue batch number(s): Lot no.: 23271

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes at room temperature, 1 hour at 37.0 ± 1.0°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

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

INTERPRETATION
Acceptability of the assay
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) In the range of 20 – 100% viability, the maximum inter-tissue variability (in viability) is ≤ 30% between two tissues treated identically.
d) In the range of 20 – 100% viability, the maximum difference in percentage between the mean viability of two tissues and one of the two tissues is ≤ 15%.

Data evaluation and statistical procedures
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: concurrent MTT reduction control

Amount/concentration applied:

36.8 to 46.9 mg of the solid test item

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

none

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute treatment

Value:

83

Negative controls validity:

valid

Remarks:

within OECD acceptable range

Positive controls validity:

valid

Remarks:

tissue viability 23%

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour treatment

Value:

89

Negative controls validity:

valid

Remarks:

within the laboratory historical control data range

Positive controls validity:

valid

Remarks:

tissue viability 8%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Norethyl was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that Norethyl did not interfere with the MTT endpoint.

The mean OD570 of the negative control tissues at the 3-minute treatment was 2.413, which is just outside the historical data range.
Evaluation: The OECD guideline indicates that values up to 2.8 are accepted, therefore this does not affect the study integrity.

The maximum inter-tissue variability in viability between two tissues treated identically was less than 16% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 9% for the negative control and test item and the positive control at the 3-minute treatment. For the positive control at the 1-hour treatment, the maximum inter-tissue variability in viability between two tissues treated identically was equal to 45% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 30%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the Norethyl is not corrosive in the in vitro skin irritation test.

Executive summary:

In an in vitro skin corrosion test using a human skin model ( EpiDerm Skin Model), the influence of the test substance on the viability of human skin was tested. Skin tissue was moistened with 25 μl of Milli-Q water and the test substance was applied directly to 0.6 cm² cultured skin (46.8 to 47.9 mg). After 3 minutes or 1 hour, the substance was removed and the viability of the tissues was tested by reduction of MTT. The positive control had a mean relative tissue viability of 21% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range at the 1-hour treatment and within the acceptable range at the 3-minute treatment. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Norethyl compared to the negative control tissues was 83% and 89%, respectively. Because the mean relative tissue viability for Norethyl was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment Norethyl is considered to be not corrosive. Based on these results Norethyl is non-corrosive in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.