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Diss Factsheets

Administrative data

Description of key information

in vitro skin corrosion: not corrosive (Wingenroth, 2016)

in vitro skin irritation: not irritating (Wingenroth, 2016)

in vitro BCOP: non corrosive to the eye (Gmelin, 2016)

in vitro HCE: not eye irritating (Wingenroth, 2016)

in vitro HETCAM: not eye irritating (Wingenroth, 2016)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: no data available
Source strain:
other: not applicable
Justification for test system used:
according to guideline
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS (CellSystems, Troisdorf, Germany)
- Tissue Lot number: 100-AE0636-1
- Date of initiation of testing: 22 Oct 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min (room temperature), 60 min (37°C)
- Temperatur used during post-treatment: after washing, incubation with MTT solution for 3 hours at 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: after the incubation period inserts were washed carefully in PBS
- Observable damage in the tissue due to washing: not applicable
- Modifications to validated SOP: not applicable

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


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Reliability of the test was previously confirmed by interlaboratory validation

NUMBER OF REPLICATE TISSUES:
All tests were performed in triplicates for each concentration and each time point

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 mg (plus 50 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)
- Concentration (if solution): undiluted


NEGATIVE CONTROL
- Amount(s) applied: 50 µl
- Concentration (if solution): 0.9% NaCl

Duration of treatment / exposure:
3 min (room temperature), 60 min (37°C)
Duration of post-treatment incubation (if applicable):
after washing, incubation with MTT solution for 3 hours at 37°C
Number of replicates:
three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Cell viability after 3 min [%]
Value:
103.41
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Cell viability after 60 min [%]
Value:
104.28
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Interpretation of results:
other: not corrosive to skin
Conclusions:
The test substance was evaluated of corrosive properties by using an artificial 3D-Skin model according to OECD guideline 431. The test substance was characterized by no significant impact on cell viability after 3 min. or after the 60 min. period. Thus, the test substance is not to be labeled as corrosive to skin.
Executive summary:

Hydroxyprogesterone was evaluated for corrosive properties by using an artificial 3D-Skin model (reconstructed human epidermis (RhE)) according to OECD guideline 431.


The test item was applied undiluted to the epiCS® skin/epidermal equivalents in triplicates and incubated 3 min. and 60 min., respectively. Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100%.
The following values of cell viability were recorded for the test item after 3 min. and after 60 min. of incubation: 103 % and 104 % (rounded), respectively. Thus, the test substance is not to be labeled as corrosive to skin.

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:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not applicable
Source strain:
other: not applicable
Justification for test system used:
commercially available test method
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Lot number: 100-AD1448-1

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 insert 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 negative control
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:
three
Irritation / corrosion parameter:
% tissue viability
Value:
92.67
Vehicle controls validity:
not applicable
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
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
Interpretation of results:
other: not irritating
Conclusions:
A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The mean value of cell viability was recorded to be 92.67%. The test item was thus shown to be not irritating to reconstructed human skin in vitro.
Executive summary:

A study was performed for the assessment of the skin irritancy of Hydroxyprogesteron with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46.


The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.
Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100% The results of the concurrent negative control (NC, 0.9 % NaCl) and positive control (PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.
The mean value of cell viability was recorded to be 92.67%. The test item was thus shown to be not irritating to reconstructed human skin in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 2015
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:
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:
Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s salt solution (HSS) with 1% penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HSS supplemented with 1% penicillin/streptomycin solution and 1 % fetal bovine serum (FBS) and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 ° C (± 1 ° C) for about 2 hours before preparation of the corneas.
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.



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:
2.2
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Based on these data, in comparison to the vehicle control, the In Vitro Irritancy Score (IVIS) value was calculated to be 2.2 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage.
Executive summary:

This study was performed to assess the corneal damage potential of the solid test item Hydroxyprogesteron with the Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea in accordance with OECD TG 437).


20 % (w/v) of the test item in physiologic saline solution (0.9 %) and the positive control imidazole in physiologic saline solution (0.9 %) were tested on 3 bovine corneas each in comparison to the negative control. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour incubation time.
Test items were applied to the epithelial surface of the cornea in the corneal holder. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea before and after treatment with the test item. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea after treatment. Based on these data, in comparison to the vehicle control, the In Vitro Irritancy Score (IVIS) value was calculated to be 2.2 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.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 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:
equivalent or similar to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
study was conducted prior to implementation of OECD TG 492
Deviations:
yes
Remarks:
0.3% SDS, in phosphate buffered saline (PBS) was used as positive control; this deviation is not considered to influence the reliability of the study
Principles of method if other than guideline:
- Principle of test:
Assessment of ocular irritation potential of the test substance by determination of cytotoxic effects (MTT assay) on a human corneal epithelium (HCE) cell model (similar to EpiOcular).
The HCE model was 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.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 μl per insert

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg/ insert plus 30 µl PBS to moisten and ensure good contact with the tissue

VEHICLE
- Amount(s) applied (volume or weight with unit): 30 µl
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. 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 %.
Irritation parameter:
mean percent tissue viability 
Value:
ca. 98.33
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Tabular Summary of the results

 

 Sample No.  Test item  OD mean*  StdDev  % Viability
 1 -3  Negative control PBS  1.05  0.10  100.00
 4 -6  Positive control SDS 0.3%  0.29  0.10  27.66
 7 -9  Hydroxyprogesteron  1.03  0.06  98.33

*6 values

Conclusions:
The reconstructed human corneal epithelial tissue-based in vitro test method (HCE; Episkin) 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 98% by the MTT conversion assay. Thus, the test item is predicted as non-irritant under the conditions of this test method.
Executive summary:

An in vitro study for assessing ocular irritation of compounds was performed with the test item Hydroxyprogesteron using a human epithelial corneal cell model. The model used is standardized and commercially available (Human Corneal Epithelial Model (HCE); Episkin, France). The study was conducted equivalent to the later implemented OECD TG 492.


Undiluted Hydroxyprogesteron was applied topically to the HCE tissue, i.e. 30 mg per insert; plus 30 μl PBS to moisten and ensure good contact with the tissue; three replicates.


After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 98 % (rounded) as measured by a MTT conversion assay.


The results of the concurrent negative (NC, PBS) and positive control (PC, SDS 0.3 %) demonstrated the viability (NC) and sensitivity (PC) of the test model.


The results show that Hydroxyprogesteron is predicted as non-irritant under the conditions of this test method.

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: "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)
Species:
other: chicken's egg
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 eggs each
- 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.
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 82 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. Undiluted test item was applied directly onto the Chorioallantoic membrane (CAM) of each egg in a volume of 300 µL (an average of 82 mg of the test item) undiluted test item ensuring that at least 50% of the CAM surface area is covered. 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 haemorrhage, vessel lysis or coagulation 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 Scrore (IS)
Run / experiment:
mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
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 membrane (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.
Executive summary:

This study was performed to assess the eye irritation potential of the test item Hydroxyprogesteron with an 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 membrane (CAM) of fertilized chicken eggs.


After the application of the test item blood vessels and albumen were examined and scored for the following irritant effects: vasodilation, slight haemorrhage; vessel lysis, strong haemorrhage; blood coagulation, albumen coagulation for a period of 300 seconds.


A volume of 300 μl of the test item (corresponding to an average of 82 mg) was applied per egg directly onto the chorioallantoic membrane.


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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

With respect to local irritant effects, a batterie of recent in vitro tests for skin irritation/corrosion (OECD TG 431 and 439) and damage to the eye (OECD TG 437, HCE, HET-CAM) is available for 17-OHP (table below). Based on these assays no corrosive or irritant potential to the skin and no damage to the eye can be concluded for the substance.


These findings are supported by in vivo data for OHPA used as source for read-across in other sections of this document. No skin irritation in rats was found for OHPA in a combined acute dermal toxicity and local tolerance study comparable to OECD TG 402 and 404 (refer also to 4.3, table) with reliability 2. In rabbits no irritation to the eye was shown for OHPA in a study according to OECD TG 405 with reliability 1 (table below).


 


Skin irritation/corrosion


Hydroxyprogesterone was evaluated for corrosive properties by using an artificial 3D-Skin model (reconstructed human epidermis (RhE)) according to OECD guideline 431.


The test item was applied undiluted to the epiCS® skin/epidermal equivalents in triplicates and incubated 3 min. and 60 min., respectively. Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100%.
The following values of cell viability were recorded for the test item after 3 min. and after 60 min. of incubation: 103 % and 104 % (rounded), respectively. Thus, the test substance is not to be labeled as corrosive to skin (Wingenroth, 2016).


 


A study was performed for the assessment of the skin irritancy of Hydroxyprogesteron with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46.


The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.
Cell viability was measured in a photometer by the amount of MTT (methylthiazole
tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100% The results of the concurrent negative control (NC, 0.9 % NaCl) and positive control (PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.
The mean value of cell viability was recorded to be 92.67%. The test item was thus shown to be not irritating to reconstructed human skin in vitro (Wingenroth, 2016).


 


Supporting data are available for the source substance hydroxyprogesterone acetate: In a combined study on acute toxicity and on local tolerance similar to OECD TG 402 HAN: WIST rats (3/sex) (3/sex) were dermally exposed to hydroxyprogesterone acetate in physiological saline for 24 hours at a limit dose of 2000 mg/kg bw under semiocclusive conditions.  Animals then were observed for 14 days.


The administration of the test substance was tolerated without mortalities, compound-related clinical findings, effects on body weight gain and gross pathological findings.


No local intolerance reactions at the application sites were observed. (Kurth, 1996)


 


Eye irritation/corrosion


A study was performed to assess the corneal damage potential of the solid test item Hydroxyprogesteron with the Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea in accordance with OECD TG 437).


20 % (w/v) of the test item in physiologic saline solution (0.9 %) and the positive control imidazole in physiologic saline solution (0.9 %) were tested on 3 bovine corneas each in comparison to the negative control. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour incubation time.
Test items were applied to the epithelial surface of the cornea in the corneal holder. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea before and after treatment with the test item. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea after treatment. Based on these data, in comparison to the vehicle control, the In Vitro Irritancy Score (IVIS) value was calculated to be 2.2 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. (Gmelin, 2016)


 


A study was performed to assess the eye irritation potential of the test item Hydroxyprogesteron with an  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 membrane (CAM) of fertilized chicken eggs.


After the application of the test item blood vessels and albumen were examined and scored for the following irritant effects: vasodilation, slight haemorrhage; vessel lysis, strong haemorrhage; blood coagulation, albumen coagulation for a period of 300 seconds.


A volume of 300 μl of the test item (corresponding to an average of 82 mg) was applied per egg directly onto the chorioallantoic membrane.


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. (Wingenroth, 2016)


 


An in vitro study for assessing ocular irritation of compounds was performed with the test item Hydroxyprogesteron using a human epithelial corneal cell model. The model used is standardized and commercially available (Human Corneal Epithelial Model (HCE); Episkin, France).


Undiluted Hydroxyprogesteron was applied topically to the HCE tissue, i.e. 30 mg per insert; plus 30 μl PBS to moisten and ensure good contact with the tissue; three replicates.


After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 98 % (rounded) as measured by a MTT conversion assay.


The results of the concurrent negative (NC, PBS) and positive control (PC, SDS 0.3 %) demonstrated the viability (NC) and sensitivity (PC) of the test model.


The results show that Hydroxyprogesteron is predicted as non-irritant under the conditions of this test method. (Wingenroth, 2016)


 


Supporting data are available for the source substance hydroxyprogesterone acetate: In a primary eye irritation study according to OECD TG 405 0.1 ml hydroxyprogesterone acetate (~61.3-70.6 mg) was instilled into the conjunctival sac of the right eye of New Zealand White rabbits (2 m + 2 f). The untreated left eye served as control. Animals were observed for 4 d.


Mainly transient slight, in one animal (no. 598M) moderate signs of irritation were observed only on the application day. From day 2 onwards, the animals were without findings. The control eyes were without findings. In his study, hydroxyprogesterone acetate was not irritating to the eye. (Treher, 1996) 


 


 


In conclusion, 17-OHP is not classified to induce skin irritation/corrosion or damage to the eye.


 


Skin and eye irritation/ corrosion studies for OHPA and 17-OHP
























































 



Hydroxyprogesterone acetate


OHPA



17-Hydroxyprogesterone


17-OHP



CAS No.



302-23-8



68-96-2



ZK



5189



5149



Skin corrosion (RhE) in vitro OECD TG 431



No data



Study no. T102383-7/ Report no. PH-39217 (Wingenroth, 2016), EpiCS


GLP, guideline study, no deviations


Compound purity 99.8%


No findings -> Not corrosive (rel-1)



Skin irritation (RhE) in vitro


OECD TG 439



No data



Study no. T102384-8/ Report no. PH-39218 (Wingenroth, 2016), EpiCS


GLP, guideline study, no deviations


Compound purity 99.8%


No findings -> Not irritant (rel-1)



Skin irr. in vivo (rat) comparable to


OECD TG 404



Comb. acute dermal & local tolerance study; refer to 4.3


TX95187/ X058; draft


(Kurth/ Treher, 1996)


GLP, guideline study with deviations


Compound purity 98.3%


No findings -> not irritant (rel-2)



No data



BCOP Eye corr./ irrit. in vitro


OECD TG 437



No data



Study no. T102496-2/ Report no. PH-39086 (Gmelin, 2016), GLP, guideline study,


Dev.: stability/ homogenicity not checked


Compound purity 99.8%


No findings -> Not corrosive (rel-1)



HETCAM Eye irrit./ corr. In vitro


 



No data



Study no. T102620-1/ Report no. PH-39222 (Wingenroth, 2016), HET-CAM model


GLP, non-guideline study


Compound purity 99.8%


No findings -> Not irritant (rel-1)



HCE Eye irrit./ corr. In vitro


 



No data



Study no. T102537-8/ Report no. PH-39214 (Wingenroth, 2016)


HCE model (EpiSkin, France)


GLP, non-guideline study


Compound purity 99.8%


No findings -> Not irritant (rel-1)



Eye corr. In vivo (rabbit),


OECD TG 405



TX95293/ X083 (draft version)


Treher, 1996;


non-GLP,  guideline study according to TG 405 (version 1987)


Compound purity 98.3%


Slight reversible findings -> Not irritant (rel-2)






No data



 

Justification for classification or non-classification

Based on the study results a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.