Estradiol

Endpoint summary

Description of key information

Skin irritation/corrosion

Skin irritation (in vitro, OECD 439): not irritating [report, Wingenroth 2016]

Skin irritation (rabbit, repeated dose dermal toxicity study): supporting; slight irritation observed which is considered to result from the occlusive patch/adhesive contained in [Hart G.E., 1992]
Eye irritation (BCOP, OECD TG 437): not corrosive [report, Gmelin 2014]

Eye irritation (HET-CAM): not classified [report, Wingenroth, 2016]

Eye irritation: (HCE): non-irritating [report, Wingenroth, 2016]

 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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 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

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)

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

27 June 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: Analytical determination of stability and homogeneity of the test item in the vehicle was not performed specifically for this study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The usage of physiologic saline solution for formulation of the test item is plausible based on the current knowledge of the sponsor. No objections were seen particularly in regard to stability.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was suspended by using a mortar shortly prior to application in physiologic saline solution, the formulation was continuously stirred.
- Final preparation of a solid: 20% (w/v)

FORM AS APPLIED IN THE TEST (if different from that of starting material): The preparation was visually described as suspension.

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- Number of animals: N/A
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transport: 1 L containers with 500 mL HSS and 1 % penicillin / streptomycin solution, transport of the containers in coolers on ice
- Indication of any existing defects or lesions in ocular tissue samples: Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin / streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.
- Selection and preparation of corneas: For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed in a cornea holder with the endothelial side on the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour into the incubator at 32 °C (± 1 °C).
- Quality check of the isolated corneas: Following 1 hour in the incubator, the MEM medium was aspirated and the chambers were refilled with fresh MEM medium. For each cornea the reference opacity value was measured then. The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean ± standard deviation were selected for the actual test and assigned to the test groups.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL per cornea and chamber
- Concentration (if solution): 20 % (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL per cornea and chamber
- Concentration (if solution): 0.9% NaCl (w/v)

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

no further incubation required

Number of animals or in vitro replicates:

3 cornea

Details on study design:

Immediately before application, the medium was aspirated from the anterior chamber. 750 μL of the test material formulations were applied to the corneas, each through the holes of the anterior chamber. The holes of both chambers were sealed with adhesive tape and the holders were kept with the front side up, so that the formulations covered the cornea sufficiently (closed chamber method). The holders were transferred into the incubator at 32 °C (± 1 °C) for the exposure time of 4 hours. After the exposure, the formulations were aspirated from the anterior chamber and the corneas were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. Before measuring opacity, fresh MEM medium was filled in the chambers and the holes were sealed with tape.

Positive control: 20 % Imidazole in physiologic saline (w/v; 750 µL)

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort 3.4 SP6 software from Testo AG, Lenzkirch). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability:The medium in anterior chamber of each holder was replaced by 1ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 μL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluoresceinsolution was prepared and also filled into the 96-well plate, in triplicates.
The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader (Bio-Tek EL 808, Software Gen5).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Decision criteria as indicated in the OECD TG 437

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

2.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Individual values of opacity, permeability and IVIS

Cornea No.	Opacity per cornea	Permeability per cornea	IVIS per cornea	IVIS per group
				mean		SD
Vehicle control                    1	-0.6	0.006	-0.5
0.9 % NaCl                              2	-0.1	0.006	0.0	-0.9		1.2
3	-2.4	0.008	-2.3
Positive Control                     4	77.3	1.004	92.3
20 % Imidazole                      5	60.5	1.122	77.4	87.1		8.4
6	75.1	1.091	91.5
Test item                                10	-1.1	0.000	-1.1
20% Estradiol           11	1.7	-0.002	1.6	2.3		3.7
12	6.2	0.005	6.3

 

Interpretation of results:

other: negative

Conclusions:

The test item (20 % (w/v) in physiologic saline) was tested in the BCOP test according to OECD TG 437. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour exposure time. Based on these data, in comparison to the vehicle control, the In Vitro Irritancy Score (IVIS) value was calculated to be 2.3 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage. The results of the positive (20 % imidazole solution) and vehicle (physiologic saline solution) controls confirmed the validity of the test system.

Executive summary:

This in vitro study was performed to assess them corneal irritation and damage potential of Estradiol (20% w/v in physiologic saline) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 27 June 2013.

The corneae were incubated with the test substance and controls for 4 h. After rinsing 3 times with phenol-red containing MEM the anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) was calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

A 20% dilution of the test substance in physiological saline caused no relevant increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 2.3.

The positive control (Imidazole 20%) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 92.3 (1st experiment) and 77.4 (2nd experiment)).

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score -0.5 (1st experiment) and 0.0 (2nd experiment)).

Since the mean in vitro irritancy score of the test substance was <55.1, a 20% dilution of Estradiol in physiological saline is considered to not be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

- Principle of test:
Assessment of ocular irritation potential of the test substance by determination of cytotoxic effects on a human corneal epithelium (HCE) cell model (similar to EpiOcular).
The HCE model is currently involved in the eye irritation validation conducted by COLIPA following ECVAM guidelines. Furthermore, it is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). This model is recognized as the model of choice and scientifically relevant as documented by several publications (e.g. Alepee et. al., Toxicology in Vitro 34 (2016) 55–70).

GLP compliance:

yes (incl. QA statement)

Species:

other: human corneal epithelial cells

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

The experiment was carried out on a Human Corneal Epithelial (HCE) Model, which is standardized and commercially available (SkinEthicTM Human Corneal Epithelial Model (HCE), SkinEthic, France). Inserts were of 0.5 cm² size. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.


- Justification of the test method (e.g. ICE, EIT, RhCE) and considerations regarding applicability: The model used is standardized and commercially available (Human Corneal Epithelial Model (HCE); Episkin, France).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: human epithelial corneal cell model, Human Corneal Epithelium (HCE/S/5); Reference HCEJ05; Episkin, France.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg per insert, plus 30 µl PBS to moisten and ensure good contact to the tissue

Duration of treatment / exposure:

60 min at room temperature

Duration of post- treatment incubation (in vitro):

16 hours in the incubator (37°C, 5% CO2, maximum humidity)

Number of animals or in vitro replicates:

three

Details on study design:

The irritation potential of the test item is assessed by determination of its cytotoxic effect on an reconstructed human ocular epithelium. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item. The test item is applied pure. Since the test item is a solution (ca. 40% in ethyl acetate) the solvent is tested in parallel to distinguish potential toxic effects of the test item from the effects of the solvent. After the post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity) MTT reduction is performed. Cell viability is measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative or positive control substances or the test item. A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50 %.


- Number of repetitions and replicates used: One repitition, triplicates
- Test chemical concentrations used: 30 mg
- Justification for choice of solvent for each test chemical: No solvent but phosphate buffered saline to moisten the substance and ensure good contact with the tissue
- Duration of exposure to the test chemical: 60 min, followed by a 16 h post incubation period.
- Description of evaluation and decision criteria used: The viability of the cells after treatment is measured using the MTT Assay. The amount of blue formazan salt is detected spectrophotmetrically at 570 nm. The negative OD values should range from ≥ 0.5 to ≤ 3.0 for solid substances. The positive control OD values should range from ≤ 50% viability of the negative control to ≤ 35% of the negative control. The difference of viability between two tissue replicates should be less than 20% or the standard deviation (SD) between three tissue replicates should not exceed 18%. The substance is considered positive if the mean tissue viability of is ≤ 50% and it does not need to be classified if the mean tissue viability is ≥ 50%

Irritation parameter:

mean percent tissue viability 

Value:

ca. 95.39

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Tabular Summary of the results

 

Sample No.	Test item	OD mean*	StdDev	% Viability
1 -3	Negative control PBS	1.11	0.05	100.00
4 -6	Positive control SDS 0.3%	0.37	0.05	32.93
7 -9	Estradiol	1.06	0.03	95.39

*6 values

Interpretation of results:

GHS criteria not met

Conclusions:

The reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted with the test item. The undiluted test item was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes followed by a 16 hours post-treatment incubation period the cell viability was measured to be about 95% by the MTT conversion assay. Thus the test item is predicted as non-irritant under the conditions of this test method.

Executive summary:

In this study conducted equivalent to OECD test guideline 492 (adopted July 28, 2015), the potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model (Skin Ethic™, HCE) from Episkin, consisting of normal, human epithelial corneal cells mimicking characteristics of the corneal epithelium.

The test item was applied topically to the HCE tissue for 60 min. After an 16 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with PBS.

For the test item no non-specific reduction of MTT and no relevant colouring after mixture with isopropanol was reported.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was ≥ 50% (95.39%).

The controls confirmed the validity of the study. The mean absolute OD570 of the negative control tissues was > 0.8 and < 2.5 (1.11). The mean relative tissue viability (% negative control) of the positive control was < 50% (32.93%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (5 %).

In this study under the given conditions the test item showed no irritant effects.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

other: Appendix B3 of "ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products", NIH Publication No. 10-7553, September 2010.

Principles of method if other than guideline:

- Principle of test: The Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) is a test method which implies the use of a complete tissue constituted of blood vessels and proteins that is capable of responding to chemical injury with an inflammatory process similar to the one occuring in the conjunctival tissue of the eye.

- Short description of test conditions: The test substance is applied directly to the chorioallantoic membrane (CAM) of fertilized chicken eggs

- Parameters analysed / observed: acute effects on haemorrhage, lysis of blood vessels and coagulation

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

SOURCE OF FERTILIZED CHICKEN EGGS
- Source: Brinkschulte Josef GmbH & Co.KG, 48308 Senden
- Number of eggs: 4
- Characteristics of donor animals fertile Lohmann Brown hens
- Treatment conditions of eggs prior initiating testing: day 1-7: an incubator with an automatic rotating device (e.g. Ehret GmbH), optimum temperature : 37.5 °C, relative humidity 63%. day 8: with the large end
upward and not rotated for ensuring accessibility to the Chorioallantoic membrane (CAM) region
- Time interval prior to initiating testing: 8 days
- indication of any existing defects or lesions in eggs: After 7 days of incubation, all eggs were candled in order to discard those that were defect and to mark the air bubble.

- Justification of the test method (e.g. ICE, EIT, RhCE) and considerations regarding applicability: According to the GUIDANCE DOCUMENT NO 263 ON INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SERIOUS EYE DAMAGE AND EYE IRRITATION SERIES ON TESTING AND ASSESSMENT Number 263, the HET-CAM test allows evaluating vascular effects. It makes use of the chorioallantoic membrane (CAM) of fertilized chicken eggs, a vascular foetal membrane composed of the fused chorion and allantois. The acute effects induced by a test chemical on the small blood vessels and proteins of this soft tissue membrane can be used as indicator of ocular effects induced by the test chemical (ICCVAM, 2010). The test method is accepted by certain countries for the identification of serious eye damage (UN GHS Cat. 1) (ECHA, 2015) although further work was recommended before a statement on its scientific validity could be made (ICCVAM 2006, 2010). One potential reason for such outcome is the existence of a variety of protocols and prediction models used for the same test method. Briefly, for the identification of serious eye damage (UN GHS Cat. 1), coagulation was the recommended endpoint based either on the mean time to develop coagulation or on the severity of coagulation observed at a single time after exposure (Spielmann et al., 1991; Steiling et al., 1999). For the identification of test chemicals not requiring classification (UN GHS No Cat.), the evaluation of coagulation, haemorrhage and lysis at different fixed time points (0.5, 2 and 5 min) was recommended (Luepke, 1985), based on the IS(a) prediction model (ICCVAM, 2010). The HET-CAM test was used to substantiate the results gathered from the OECD 437 test.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 300 μL/egg which corresponded to an amount from an average of 95 mg.

Duration of treatment / exposure:

300 sec

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

4 eggs

Details on study design:

At day 8 of incubation the sections marked for the air bubble were sawed out of the shell. The inner membrane was moistened with NaCl 0.9 % and carefully removed with forceps. Only eggs with normally developed embryos and blood vessel systems were used for testing. Undilutet test item was applied directly onto the Chorioallantoic membrane (CAM) of each egg in a volume of 300 µL undiluted test item. 4 eggs each were used for the test item, negative and positive controls.

Observations of effects to the blood vessels, albumen or embryo over a period of 300 seconds after substance application are determined for each single egg. The time to the appearance of each of the observations mentioned above has been monitored and recorded. If no effect appeared during the observation period of 300 seconds (observation = 0) the result was assigned as negative for the related endpoint, and the factor set to 0 for this endpoint when calculating the Irritation Score (IS).

Scoring criteria for the acute effects and calculation of Irritancy Score (IS):

0 = no effect
1 = vasodilatation, slight haemorrhage (H)
2 = vessel lysis, strong haemorrhage (L)
3 = blood-coagulation, albumen-coagulation (C)

IS = 5 x (301-sec H)/300 + 7 x (301- sec L)/300 + 9 x (301- sec C)/ 300

H= observed start in seconds of haemorrhage reactions; L= observed start in seconds of vessel lysis, strong haemorrhage; C= observed start in seconds of blood - coagulation, albumen - coagulation

Data Interpretation:

Irritation Score (IS)

0-0.9 -> Non irritant
1-4.9 -> Slight irritant
5-8.9 -> Moderate irritant
9-21 -> Strong irritant

A test substance is considered to cause severe irritation when the IC value is greater than nine.

Irritation parameter:

other: irritation score (IS)

Run / experiment:

egg #1-#4

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A test substance is considered to cause severe irritation when the IC value is greater than nine.

Table 1: Summary of results, test item

Egg	Effect	Effect detected after [sec]	Irritation Score (IS)
9	1	> 300
	2	> 300
	3	> 300	0
10	1	> 300
	2	> 300
	3	> 300	0
11	1	> 300
	2	> 300
	3	> 300	0
12	1	> 300
	2	> 300
	3	> 300	0

effect: 1 = vasodilatation, slight haemorrhage 2 = vessel lysis, strong haemorrhage 3 = blood-coagulation, albumen-coagulation

Interpretation of results:

other: negative

Conclusions:

The test item was tested in the in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method. This is a method that makes use of the chorioallantoic mambrane (CAM) of fertilized chicken eggs. For determination of acut effects on haemorrhage, lysis of blood vessels and coagulation the undiluted test item was directly applied onto the CAM for 5 minutes. The Irritation Score (IS) value was calculated to be 0 for the test item which was interpreted as being non irritant. The results of the positive ( SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system.

Executive summary:

The test item was tested in the in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method. This is a method that makes use of the chorioallantoic mambrane (CAM) of fertilized chicken eggs for determination of acut effects on haemorrhage, lysis of blood vessels and coagulation the undiluted test item was directly applied onto the CAM for 5 minutes. Subsequently, the Irritation Score (IS) is measured for the negative control (0.9 % NaCl) the positive control (1% SDS) and the test item. The Irritation Score (IS) value was calculated to be 0 for the test item which was interpreted as being non irritant. The results of the positive ( SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system, i.e. a mean IS of 14 for the positive control and a mean IS of 0 for the negative control. The results of this test substantiate the results gathered form the test conducted according to OECD 437, that the substance does not need to be classified according to Regulation (EU) No. 1272/2008 (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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.

 

In a 28 day irritation study rabbits were treated with 3M estradiol TDD patch containing 1.57 mg estradiol. The patches were applied to abraded and intact skin once weekly for a 7 day wear interval for 3 weeks to a group of 6 rabbits (3/sex/group) followed by a four week observation period. After removal the skin was evaluated for irritation at 1h, 24h, 48h, 72h and 7 days after removal. Blood samples were taken 0h, 4h, 8h, 24h, 32h, 48h and 72h during first week of dosing for analysis of estradiol.

No apparent difference between abraded and intact skin or between placebo and estradiol containing patch could be observed.
Slight and reversible irritation was provoked by the 3M estradiol TDD patch.
Irritation was rather due to adhesion of patches than of test substance estradiol.
Classification of estradiol is not required.

 

An in vitro study was performed to assess them corneal irritation and damage potential of Estradiol (20% w/v in physiologic saline) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 27 June 2013.

The corneae were incubated with the test substance and controls for 4 h. After rinsing 3 times with phenol-red containing MEM the anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) was calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

A 20% dilution of the test substance in physiological saline caused no relevant increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 2.3.

The positive control (Imidazole 20%) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 92.3 (1st experiment) and 77.4 (2nd experiment)).

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score -0.5 (1st experiment) and 0.0 (2nd experiment)).

Since the mean in vitro irritancy score of the test substance was <55.1, a 20% dilution of Estradiol in physiological saline is considered to not be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

 

The test item was tested in the in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method. This is a method that makes use of the chorioallantoic mambrane (CAM) of fertilized chicken eggs for determination of acut effects on haemorrhage, lysis of blood vessels and coagulation the undiluted test item was directly applied onto the CAM for 5 minutes. Subsequently, the Irritation Score (IS) is measured for the negative control (0.9 % NaCl) the positive control (1% SDS) and the test item. The Irritation Score (IS) value was calculated to be 0 for the test item which was interpreted as being non irritant. The results of the positive ( SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system, i.e. a mean IS of 14 for the positive control and a mean IS of 0 for the negative control. The results of this test substantiate the results gathered form the test conducted according to OECD 437, that the substance does not need to be classified according to Regulation (EU) No. 1272/2008 (CLP).

 

 

In a study conducted equivalent to OECD test guideline 492 (adopted July 28, 2015), the potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model (Skin Ethic™, HCE) from Episkin, consisting of normal, human epithelial corneal cells mimicking characteristics of the corneal epithelium.

The test item was applied topically to the HCE tissue for 60 min. After an 16 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with PBS.

For the test item no non-specific reduction of MTT and no relevant colouring after mixture with isopropanol was reported.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was ≥ 50% (95.39%).

The controls confirmed the validity of the study. The mean absolute OD570 of the negative control tissues was > 0.8 and < 2.5 (1.11). The mean relative tissue viability (% negative control) of the positive control was < 50% (32.93%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (5 %).

In this study under the given conditions the test item showed no irritant effects.

Justification for classification or non-classification

Due to the results of the described studies no classification is required according to Regulation (EC) 1272/2008/EC (CLP), Annex I.