[(8R,9S,10R,13S,14S,17R)-3-ethoxy-17-ethynyl-13-methyl-2,7,8,9,10,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthren-17-yl] acetate

Endpoint summary

Description of key information

In two reliable in vitro skin corrosion and skin irritation studies performed with a structural analogue of Norethylac, Norethyl, the test substance was not irritating and not corrosive to skin. This result can be read across to Norethylac.

In a reliable Bovine Corneal Opacity and Permeability (BCOP) assay performed with Norethyl, the test substance was not irritating to eyes. This result can be read across to Norethylac.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2015 - March 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: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on animal used as source of test system:

Test system
EpiDerm Skin Model (EPI-200, Lot no.: 23271 kit EE and FF).
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Source
MatTek Corporation, Ashland MA, U.S.A.

Justification for test system used:

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

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200, kit EE and FF).
- Tissue batch number(s): Lot no.: 23271

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes at room temperature, 1 hour at 37.0 ± 1.0°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

INTERPRETATION
Acceptability of the assay
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) In the range of 20 – 100% viability, the maximum inter-tissue variability (in viability) is ≤ 30% between two tissues treated identically.
d) In the range of 20 – 100% viability, the maximum difference in percentage between the mean viability of two tissues and one of the two tissues is ≤ 15%.

Data evaluation and statistical procedures
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: concurrent MTT reduction control

Amount/concentration applied:

36.8 to 46.9 mg of the solid test item

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

none

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute treatment

Value:

83

Negative controls validity:

valid

Remarks:

within OECD acceptable range

Positive controls validity:

valid

Remarks:

tissue viability 23%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour treatment

Value:

89

Negative controls validity:

valid

Remarks:

within the laboratory historical control data range

Positive controls validity:

valid

Remarks:

tissue viability 8%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Norethyl was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that Norethyl did not interfere with the MTT endpoint.

The mean OD570 of the negative control tissues at the 3-minute treatment was 2.413, which is just outside the historical data range.
Evaluation: The OECD guideline indicates that values up to 2.8 are accepted, therefore this does not affect the study integrity.

The maximum inter-tissue variability in viability between two tissues treated identically was less than 16% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 9% for the negative control and test item and the positive control at the 3-minute treatment. For the positive control at the 1-hour treatment, the maximum inter-tissue variability in viability between two tissues treated identically was equal to 45% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 30%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the Norethyl is not corrosive in the in vitro skin irritation test.

Executive summary:

In an in vitro skin corrosion test using a human skin model ( EpiDerm Skin Model), the influence of the test substance on the viability of human skin was tested. Skin tissue was moistened with 25 μl of Milli-Q water and the test substance was applied directly to 0.6 cm² cultured skin (46.8 to 47.9 mg). After 3 minutes or 1 hour, the substance was removed and the viability of the tissues was tested by reduction of MTT. The positive control had a mean relative tissue viability of 21% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range at the 1-hour treatment and within the acceptable range at the 3-minute treatment. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Norethyl compared to the negative control tissues was 83% and 89%, respectively. Because the mean relative tissue viability for Norethyl was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment Norethyl is considered to be not corrosive. Based on these results Norethyl is non-corrosive in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Januray 2016 - March 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)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on animal used as source of test system:

Test system
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 16-EKIN-007).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Source
SkinEthic Laboratories, Lyon, France.

Justification for test system used:

Norethyl has been tested previously in the Skin corrosion test using EpiDerm as a skin model and was found not corrosive (project 511448). The objective of this study was to evaluate Norethyl for its ability to induce skin irritation using Episkin as a skin model. EpiDerm and Episkin are recommended for testing skin corrosion and skin irritation, respectively. For this purpose Norethyl was topically applied on a human three dimensional epidermal model.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin irritation potential of a test item by assessment of its effect on a three dimensional human epidermis model (1-10).
The test consists of topical application of Norethyl on the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is performed.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: concurrent MMT reduction control

Amount/concentration applied:

14.0 to 18.9 mg Norethyl, in presence of 5 μl Milli-Q water

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 15 minutes

Value:

94

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Acceptability of the assay
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Data evaluation and statistical procedures
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the test substance is not irritating in the in vitro skin irritation test.

Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm² cultured skin (at least 10 mg, in presence of 5 μl Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 10% whereas the test substance showed cell viability of 94%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a structural analogue

Justification for type of information:

The read-across rationale is attached in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".

Version / remarks:

31 May 2008

Deviations:

no

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute treatment

Value:

83

Negative controls validity:

valid

Remarks:

within OECD acceptable range

Positive controls validity:

valid

Remarks:

tissue viability 23%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour treatment

Value:

89

Negative controls validity:

valid

Remarks:

within the laboratory historical control data range

Positive controls validity:

valid

Remarks:

tissue viability 8%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Norethyl was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that Norethyl did not interfere with the MTT endpoint.

The mean OD570 of the negative control tissues at the 3-minute treatment was 2.413, which is just outside the historical data range.
Evaluation: The OECD guideline indicates that values up to 2.8 are accepted, therefore this does not affect the study integrity.

The maximum inter-tissue variability in viability between two tissues treated identically was less than 16% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 9% for the negative control and test item and the positive control at the 3-minute treatment. For the positive control at the 1-hour treatment, the maximum inter-tissue variability in viability between two tissues treated identically was equal to 45% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 30%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin irritation test with a structural analogue of Norethylac, Norethyl, was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the Norethyl is not corrosive in the in vitro skin irritation test. This result can be read across to Norethylac.

Executive summary:

In an in vitro skin corrosion test using a human skin model ( EpiDerm Skin Model), performed with a structural analogue of Norethylac, Norethyl, the influence of the test substance on the viability of human skin was tested. Skin tissue was moistened with 25 μl of Milli-Q water and the test substance was applied directly to 0.6 cm² cultured skin (46.8 to 47.9 mg). After 3 minutes or 1 hour, the substance was removed and the viability of the tissues was tested by reduction of MTT. The positive control had a mean relative tissue viability of 21% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range at the 1-hour treatment and within the acceptable range at the 3-minute treatment. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Norethyl compared to the negative control tissues was 83% and 89%, respectively. Because the mean relative tissue viability for Norethyl was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment Norethyl is considered to be not corrosive. Based on these results Norethyl is non-corrosive in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations. This result can be read across to Norethylac.

Reference 4

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a structural analogue

Justification for type of information:

The read-across rationale is attached in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

20 July 2012

Deviations:

no

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 15 minutes

Value:

94

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Acceptability of the assay
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Data evaluation and statistical procedures
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin irritation test with a structural analogue of Norethylac, Norethyl, was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the test substance is not irritating in the in vitro skin irritation test. This result can be read across to Norethylac.

Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), performed with a structural analogue of Norethylac, Norethyl, the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm² cultured skin (at least 10 mg, in presence of 5 μl Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 10% whereas the test substance showed cell viability of 94%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations. This result can be read across to Norethylac.

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:

December 2015 - March 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:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

December 2010

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System:
Bovine eyes were used as soon as possible after slaughter.

Rationale:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Transport:
Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Amount / concentration applied:

TEST MATERIAL
- 307.7 to 333.9 mg

NEGATIVE CONTROL
- 750 µl of physiological saline per cornea

POSITIVE CONTROL
- 750 µl 20% (w/v) Imidazole per cornea

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

none

Number of animals or in vitro replicates:

3 replicates were used

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
Opacity determinations will be performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea will be read against a cMEM filled chamber, and the initial opacity reading thus determined will be recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

POSITIVE CONTROL USED
20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

Vehicle
A solubility test in physiological saline was performed. Since no workable suspension of Norethyl in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies).
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microplate reader (TECAN Infinite® M200 Pro Plate Reader) (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

Acceptability of the assay
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:

in vitro irritation score

Value:

-1.1

Negative controls validity:

valid

Remarks:

IVIS -0.9

Positive controls validity:

valid

Remarks:

IVIS 113.4

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from -2.6 to 0.6. The individual positive control in vitro irritancy scores ranged from 109 to 121. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with Norethyl showed opacity values ranging from -2.8 to -1.0 and permeability values ranging from -0.005 to 0.144. The corneas were clear after the 240 minutes of treatment with Norethyl. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.8 to 0.5 after 240 minutes of treatment with Norethyl.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 113 and within two standard deviations of the current historical positive control mean (APPENDIX 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

Since Norethyl induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The eye hazard potential of Norethyl was assessed using the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD guidelines and GLP principles. The eye damage of Norethyl was tested through topical application for approximately 240 minutes. The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 113 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Norethyl did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.1 after 240 minutes of treatment. Since Norethyl induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a structural analogue

Justification for type of information:

The read-across rationale is attached in section 13.

Reason / purpose for cross-reference:

read-across source

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:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

December 2010

Deviations:

no

Irritation parameter:

in vitro irritation score

Value:

-1.1

Negative controls validity:

valid

Remarks:

IVIS -0.9

Positive controls validity:

valid

Remarks:

IVIS 113.4

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from -2.6 to 0.6. The individual positive control in vitro irritancy scores ranged from 109 to 121. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with Norethyl showed opacity values ranging from -2.8 to -1.0 and permeability values ranging from -0.005 to 0.144. The corneas were clear after the 240 minutes of treatment with Norethyl. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.8 to 0.5 after 240 minutes of treatment with Norethyl.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 113 and within two standard deviations of the current historical positive control mean (APPENDIX 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The BCOP assay was conducted with a structural analogue of Norethylac, Norethyl. Since Norethyl induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage. This result can be read across to Norethylac.

Executive summary:

The eye hazard potential of a structural analogue of Norethylac, Norethyl, was assessed using the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD guidelines and GLP principles. The eye damage of Norethyl was tested through topical application for approximately 240 minutes. The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 113 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Norethyl did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.1 after 240 minutes of treatment. Since Norethyl induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage. This result can be read across to Norethylac.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), performed with a structural analogue of Norethylac, Norethyl, the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm²cultured skin (at least 10 mg, in presence of 5 μl Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 10% whereas the test substance showed cell viability of 94%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations. This result can be read across to Norethylac.

In an in vitro skin corrosion test using a human skin model ( EpiDerm Skin Model), performed with a structural analogue of Norethylac, Norethyl, the influence of the test substance on the viability of human skin was tested. Skin tissue was moistened with 25 μl of Milli-Q water and the test substance was applied directly to 0.6 cm²cultured skin (46.8 to 47.9 mg). After 3 minutes or 1 hour, the substance was removed and the viability of the tissues was tested by reduction of MTT. The positive control had a mean relative tissue viability of 21% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range at the 1-hour treatment and within the acceptable range at the 3-minute treatment. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Norethyl compared to the negative control tissues was 83% and 89%, respectively. Because the mean relative tissue viability for Norethyl was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment Norethyl is considered to be not corrosive. Based on these results Norethyl is non-corrosive in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations. This result can be read across to Norethylac.

The eye hazard potential of a structural analogue of Norethylac, Norethyl, was assessed using the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD guidelines and GLP principles.The eye damage of Norethyl was tested through topical application for approximately 240 minutes.The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas.The meanin vitroirritancy score of the positive control (20% (w/v) Imidazole) was 113 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Norethyl did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.1 after 240 minutes of treatment. Since Norethyl induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage. This result can be read across to Norethylac.

Justification for classification or non-classification

Based on the results of two reliable in vitro skin corrosion and skin irritation studies performed with a structural analogue of Norethylac, Norethyl, classification of Norethylac for skin irritation/corrosion is not warranted in accordance with Regulation (EC) 1272/2008.