Estradiol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2015

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Estradiol

EC Number:

200-023-8

EC Name:

Estradiol

Cas Number:

50-28-2

Molecular formula:

C18H24O2

IUPAC Name:

estra-1,3,5(10)-triene-3,17-diol

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Commercially available test method.
Regulation (EU) No. 1907/2006 (REACH) Annex VII section 8.2.1 requires first testing of serious eye damage in an in vitro test system. The test method was chosen in order to identify whether the substance is corrosive to the eye or does not need a classification.

Because systemic reactions play a minor role in modulating local skin toxicity potential of
chemicals, skin irritation potential may be predicted by in vitro systems, provided they are
sufficiently complex to mimic human skin barrier and cell reactivity. The test consists of a
topical exposure of the neat substance to a human reconstituted epidermis model followed by
a cell viability test (MTT-assay).
There are several synonyms for artificial 3D-skin models like human skin recombinants,
reconstructed human skin/epidermis, 3D-human skin/epidermal equivalents, in vitro
engineered skin/epidermal substitutes, artificial skin/epidermis. The 3D-skin used closely
mimics the biochemical and physiological properties of the upper part of the human skin.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: the optical density of the isopropanol-extracts of 3 inserts was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA:

- The mean optical density (OD) values obtained with the test item were used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg (plus 30 µL 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 0.9% NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS in physiological saline

Duration of treatment / exposure:

20 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicate

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 99

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- DEMONSTRATION OF TECHNICAL PROFICIENCY:

Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, Mean OD of the tissue replicates treated with the negatice control is between > 0.8 and < 2.8 (2.23)
- Acceptance criteria met for positive control: Yes, the mean viability of the tissue replicates treated with the positive control, expressed as % of negative control is < 20% (1.38)
- Acceptance criteria met for variability between replicate measurements: Yes, standard deviation between tissue replicates is < 18% (StDev: 0.12)

Any other information on results incl. tables

Table 1: Tabular Summary of the results

Sample No.	Test item	OD mean*	StdDev	% Viability
1-3	Control NaCl 0.9 %	2.23	0.12	100.00
4-6	Positive control SDS 5 %	0.03	0.01	1.38
7 -9	Estradiol, roh (EP)	2.21	0.12	99.17

* 6 values

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Estradiol was not irritating in the in vitro skin irritation test under the experimental conditions decribed in this report.

Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), Estradiol was applied to the three-dimensional human epidermis model tissue for an exposure period of 20 minutes in triplicates. 30 μL of 0.9% NaCl were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 30 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

 

After 20 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

The positive (5% SDS) and negative (0.9% NaCl) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

 

The relative mean tissue viability obtained after 20 minutes treatment with Estradiol compared to the negative control tissues was 99%. Since the mean relative tissue viability for the test substance was above 50%, Estradiol is identified to be not irritating.