(8R,9S,10R,13S,14S,17R)-17-ethynyl-17-hydroxy-13-methyl-1,2,8,9,10,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 January 2018 - 12 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142,

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(1R,3aS,3bR,9aR,9bS,11aS)-1-ethynyl-1-hydroxy-11a-methyl-1H,2H,3H,3aH,3bH,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-one

EC Number:

608-630-3

Cas Number:

31528-46-8

Molecular formula:

C20H24O2

IUPAC Name:

(1R,3aS,3bR,9aR,9bS,11aS)-1-ethynyl-1-hydroxy-11a-methyl-1H,2H,3H,3aH,3bH,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-one

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

Justification for test system used:

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

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 27667
- The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0ºC.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.0-37.3°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after exposure and after incubation tissues were washed with phosphate buffered saline (once)
- Observable damage in the tissue due to washing: no

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

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.708 +/- 0.053
- Barrier function: 8.47 hours
- Contamination: sterile (no contamination)

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment with 3 independent OD570 measurements per replicate.

ACCEPTABILITY CRITERIA
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
- 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:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
- 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

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 26.20 - 27.44 mg
- The test item was applied directly on top of the skin tissues (pre-wetted with 25 μl Milli-Q water)

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours in MTT medium

Number of replicates:

2 tissues for the test item, 2 for the negative control and 2 for the positive control for both the 3-minute and 1-hour time point

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 2 replicates

Run / experiment:

3-minute exposure

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean tissue viability: 15%

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 2 replicates

Run / experiment:

1-hour exposure

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean tissue viability: 8.9%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: the results of the positive control data were within the historical data range and thereby showing the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was within the laboratory historical control data range.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1-hour exposure to the positive control was <15 % (i.e., 8.9%).
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation (CV) between tissue replicates was ≤ 30% (i.e., ≤ 27%)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) 1272/2008

Conclusions:

In the in vitro skin corrosion test, D6-Noreth was found to be not corrosive to the skin (mean tissue viability of 101% and 104% after a 3-minute and a 1-hour exposure period, respectively). The test item is not classified according to GHS and according to Regulation (EC) 1272/2008.