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Administrative data

Description of key information

in vitro skin irritation: not irritating (Wingenroth, 2017)

in vitro skin corrosion: not corrosive (Wingenroth, 2017)

in vitro eye irritation (BCOP): not corrosive (Gmelin, 2017)

in vitro eye irritation (EpiOc): not irritating (Wingenroth, 2017)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
August 2016
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)
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-AE2014-1

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 %.
- Thus the test item is identified as irritant and requiring classification 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:
109.15
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
Executive summary:

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 109.15%. The test item was thus shown to be not irritating to reconstructed human skin in vitro.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
August 2016
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)
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-AE2014-1


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

Positive control:
- Amount(s) applied: 50 µl
- Concentration (if solution): 4N NaOH

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:
103.51
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:
99.74
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
other: not corrosive to skin
Executive summary:

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.

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:
weight of evidence
Study period:
Sep 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)
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 shortly prior to application in isotonic saline solution to achieve
a concentration of 20% (w/v).
- 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:
0.7
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Interpretation of results:
other: non corrosive
Executive summary:

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 0.7 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:
weight of evidence
Study period:
October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according 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:
July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: reconstucted human cornea-like epithelium (RhCE)
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcular™ eye irritation test (EIT) follows international OECD test guidelines. The EIT measures the ocular irritation potential of a test item by determination of cytotoxic effects on a reconstructed human cornea epithelium (RhCE) tissue model to discriminate chemicals not requiring classification for eye irritancy (UN GHS No Category) from those requiring classification. The EpiOcular™ EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell line
The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The model is standardized and commercially available. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier (MatTek Corporation, Slovakia).
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): 50 μg per tissue insert in duplicate (for liquids: 50 μL
per tissue insert in duplicate)
- Concentration (if solution): neat test item
POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 μL methyl acetate per tissue insert in duplicate
- Concentration (if solution): neat
- Lot/batch no. (if required): 091916MHB
- Purity: 99.0 %
Duration of treatment / exposure:
6 hours under cell culture conditions (at 37°± 2°C, 5% CO2 and maximal humidity in an incubator) followed by a post-soak immersion period of about 25 min
Duration of post- treatment incubation (in vitro):
18 hours under cell culture conditions
Number of animals or in vitro replicates:
tissue inserts were used in duplicate for test item, negative and positive control
Details on study design:
RhCE tissue construct used, including batch number: EpiOcular RhCE tissue supplied by MatTek Corporation, Lot-No. 23742

Pre-check for potential optical interferences of the test item:
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability. The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment of potential direct MTT-reduction of the test item
In case of a direct MTT-reduction of the test item a killed tissue control (inserts, which were killed by freezing) was used in the main assay
2. Assessment of potential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol
In case of an influence of test item color on OD measurement, a color control was used in the main assay.
The evaluation criteria for the pre-check were:
- For MTT reduction (visual assessment): If the MTT solution color turns blue/purple, the test item is presumed to have reduced the MTT. A killed control must be conducted.
- For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is >0.08 a Color Control must be conducted.

Procedure for killed control or color control:
Killed control and color control not required.

- Description of the method used to quantify MTT formazan
For viability testing the inserts were placed in new plates containing MTT solution (1 mg/ml in Maintenance medium at 37°C, or Assay medium for the color control inserts). The tissues were incubated for 180 ± 10 min. under cell culture conditions. The extraction of blue formazan was performed in isopropanol on a vertical shaker for 2-3 hours at room temperature. The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer (EL808, Bio-Tek; 96 well format, 200 μl). Data acquisition and evaluation were performed with the software "Gen5" (Bio-Tek).


- Assay acceptance criteria:
The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.5
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.
Irritation parameter:
other: final cell viability in %
Value:
111
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:

According to the conducted pre-check the test item was demonstrated not to exert optical interferences directly affecting the test results that are not related to cytotoxic effects on tissue cells. Therefore no killed control and no color control had to be conducted.
Interpretation of results:
GHS criteria not met
Executive summary:

An in vitro study for assessing ocular irritation properties of the test item was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcularTM. The model used is standardized and commercially available. The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period. Cell viability was measured in a spectrophotometer by assessing the extent 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 was set at 100 %. The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model. The final mean percent tissue viability recorded for the test item is 111 % (rounded).

According to the results of this study the test item Gestonoronacetat was identified as not requiring classification for eye irritation according to UN GHS (No Category).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

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 109.15%. The test item was thus shown to be not irritating to reconstructed human skin in vitro.

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.

Eye irritation:

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 0.7 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.

An in vitro study for assessing ocular irritation properties of the test item was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcularTM. The model used is standardized and commercially available. The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period. Cell viability was measured in a spectrophotometer by assessing the extent 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 was set at 100 %. The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model. The final mean percent tissue viability recorded for the test item is111 % (rounded).

According to the results of this study the test item Gestonoronacetat was identified as not requiring classification for eye irritation according to UN GHS (No Category).

Justification for classification or non-classification

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