Registration Dossier

Administrative data

Description of key information

Skin irritant, Cat. 2

Not classified for skin corrosion

Eye irritant, Cat. 2

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:
key study
Study period:
22 August 2017 - 28 August 2017
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 (incl. QA statement)
Remarks:
dd. 03 November 2015
Specific details on test material used for the study:
- No correction factor for purity required.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
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).
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
- EPISKIN Small Model (TM) (EPISKIN-SM (TM), 0.38 cm^2, Lot no.: 17-EKIN-034); a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen and cultured for 13 days.

SKIN DISC PREPARATION
- Procedure used: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 21 hours at 37°C. No further preparation was applied.

ENVIRONMENTAL CONDITIONS
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.3-37.4 °C
- Humidity (%): 66-93

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1, with phosphate buffered saline
- Observable damage in the tissue due to washing: no

MTT INTERACTION
Interaction with MTT was tested in a previous conducted skin corrosion test, it was concluded that the test item did not interfere with the MTT endpoint.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Although the test item did not interfere with MTT, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity valuation with MTT. In addition to the normal procedure, one tissue was treated with test item. Instead of MTT solution this tissue was incubated with assay medium.
- Procedure used to prepare the killed tissues: Living epidermis was transferred to 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.
The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin 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.
- The test substance is considered to be non-irritant to skin 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.

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item.

ACCEPTABILITY CRITERIA:
-  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.
- 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.
- The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
- The %NSCliving should be ≤ 30% relative to the negative control OD.
- The non-specific MTT reduction should be ≤ 30% relative to the negative control OD.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 11.64 - 15.98 mg
- The test item was applied directly to the tissues (moistened with 5 μL Milli-Q water)

NEGATIVE CONTROL
- Amount applied: 25 μL PBS

POSITIVE CONTROL
- Amount applied: 25 μL 5% SDS
- The positive control was respread after 7 minutes of contact time.
Duration of treatment / exposure:
15 ±0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours and 3 hours with MTT
Number of replicates:
- 3 tissues for the test item
- 3 tissues for the positive and the negative controls each
- 3 killed tissues treated with test item and 3 killed untreated tissues
Irritation / corrosion parameter:
% tissue viability
Value:
15
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
15 ± 6.7%
Remarks on result:
other: SD: 5.6%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: the mean tissue viability of the positive controls was 15%, showing that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control was within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability was ≤ 18 (i.e., 5.3).
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was ≤ 50% relative to the negative control (i.e., 15%) and the Standard Deviation value (SD) of the % viability was ≤ 18 (i.e., 6.7)
- Acceptance criteria met for variability between replicate measurements: yes, the SD calculated from individual % tissue viabilities of the three identically treated was ≤ 18 (i.e., 5.6)
- The %NS MTT: -0.301

- Since the test item showed to be not corrosive to the skin in an earlier study, the test item is classified as Category 2 taken the results of both the corrosion and the irritation study into account.

Table 2 Individual OD measurements at 570 nm

 

A

(OD570)

B

(OD570)

C

(OD570)

Negative control

OD570measurement 1

OD570measurement 2

0.9943

1.0347

0.9044

0.9579

0.9697

1.0724

Menadione on viable tissue

OD570measurement 1

OD570measurement 2

0.1342

0.1460

0.2585

0.2523

0.1540

0.1527

Menadione on killed tissue

OD570measurement 1

OD570measurement 2

0.1322

0.1413

0.1463

0.1445

0.1597

0.1606

Non treated killed tissue

OD570measurement 1

OD570measurement 2

0.1562

0.1625

0.1454

0.1623

0.1377

0.1376

Positive control

OD570measurement 1

OD570measurement 2

0.2312

0.2329

0.2012

0.2064

0.1277

0.1298

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Table 3 Historical data

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Positive control

(viability; %)

Range

0.676 – 1.336

0.036 – 0.549

2.85 – 45.43

Mean

1.01

0.16

15.74

SD

0.16

0.10

9.22

n

155

154

163

SD = Standard deviation; n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Category 2 according to Regulation (EC) 1272/2008
Conclusions:
An in vitro skin irritation study showed that Menadione was irritant to the skin (mean tissue viability of 15%) and should be classified as Category 2 according to GHS and according to Regulation (EC) 1272/2008.
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 July 2017 - 14 July 2017
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:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- No correction factor for purity required.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 26490
- Surface: 0.6 cm^2
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator.
- Test for color interference by the test item: 25 mg test item was added to 0.3 mL Milli-Q water and the mixture was incubated for approx. 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue/purple color change was observed. A negative control (Milli-Q water) was tested concurrently.
- Test for reduction of MTT by the test item: 25 mg test item was added to 1mL MTT solution (1mg/mL in phosphate buffered saline. The mixture was incubated for approx. 1 hour at 37.0 ± 1.0°C. At the end of the exposure time it was checked if a blue/purple color change was observed or a blue/purple precipitate was observed. A negative control (Milli-Q water) was tested concurrently.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.8-37.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after exposure and after incubation tissues were washed with phosphate buffered saline (once)
- Observable damage in the tissue due to washing: no

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 REPLICATE TISSUES: 2 per exposure duration + 2 for the negative control and the positive control each

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment with 3 independent OD570 measurements per replicate.

ACCEPTABILITY CRITERIA
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range (≥0.8 and ≤2.8).
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
- 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:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
- 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
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25.1 - 36.3 mg
- The test item was applied directly on top of the skin tissues (pre-wetted with 25 μl Milli-Q water), spread to match the size of the tissue.

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL (8N KOH)

Duration of treatment / exposure:
3 minute and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours in MTT medium
Number of replicates:
2 tissues per test item per exposure time (4 tissues in total)
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 replicates
Run / experiment:
3-minute exposure
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
mean tissue viability: 6.5%
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 replicates
Run / experiment:
1-hour exposure
Value:
81
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
mean tissue viability: 11%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: the results of the positive control data were within the historical data range and thereby showing the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was within the laboratory historical control data range (i.e., 1.718 for the 3-minute exposure period and 1.490 for the 1-hour exposure period).
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1-hour exposure to the positive control was <15 % (i.e., 11%).
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation (CV) between tissue replicates was ≤ 30% (i.e., ≤ 22%)

Table 1 Individual OD measurements at 570 nm

 

3-minute application (OD570)

       A               B

1-hour application (OD570)

       A               B

Negative control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

 

1.6989

1.7387

1.2470

1.6792

1.8178

1.7863

1.4028

1.7471

1.7492

1.7720

1.3922

1.7311

Menadione

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

 

1.7633

1.7594

1.1644

1.2748

1.9101

1.6178

1.2889

1.2677

1.8572

1.6556

1.2826

1.2631

Positive control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

 

0.1498

0.1506

0.1906

0.2170

0.1630

0.1508

0.2064

0.2273

0.1629

0.1516

0.2039

0.2269

OD = Optical density

Duplicate exposures are indicated by A and B.

Table 2 Historical control data

 

Negative control

Positive control

Positive control

 

3-minute treatment

(OD570)

1-hour treatment

(OD570)

3-minute treatment

(OD570)

1-hour treatment

(OD570)

3-minute treatment

(% viability)

1-hour treatment

(% viability)

Range

1.324 – 2.615

1.361 – 2.352

0.0172 – 0.56

0.046 – 0.339

6 – 25

3 – 13

Mean

1.84

1.85

0.19

0.14

11.03

7.45

SD

0.26

0.22

0.09

0.06

4.39

2.51

n

81

83

80

77

38

38

SD = Standard deviation; n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016.

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) 1272/2008.
Conclusions:
In the in vitro skin corrosion test, Menadione was found to be not corrosive to the skin (mean tissue viability of 100% and 81% after a 3-minute and a 1-hour exposure period, respectively). The test item is not classified according to GHS and according to Regulation (EC) 1272/2008.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
02 August 2017 - 04 August 2017
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:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
dd 03 November 2015
Specific details on test material used for the study:
No correction for purity required
Species:
human
Details on test animals or tissues and environmental conditions:
TEST SYSTEM:
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
- Justification: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 55.7 - 65.3 mg
- The test material was directly applied to the tissues which were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS.
- Positive control: 50 μl Methyl Acetate
- Negative control: 50 μl Milli-Q water
Duration of treatment / exposure:
6 hours ± 15 minutes
Duration of post- treatment incubation (in vitro):
Post-soak: 25 ± 2 minutes; incubation after the post-soak: 18 hours ± 15 minutes
Number of animals or in vitro replicates:
2 tissues per test item and 2 tissues for the negative and the positive control each.
Details on study design:
- Details of the test procedure used:
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23491)
- Duration and temperature:
* exposure: 6 hours ± 15 minutes at 37.0 ± 1.0°C
After the exposure period, the tissues were thoroughly rinsed with Ca2+Mg2+-free DPBS (brought to room temperature) to remove residual test item.
* post-soak immersion: 25 ± 2 minutes at room temperature in assay medium
* incubation: 18 hours ± 15 minutes at 37°C in assay medium + 180 minutes with MTT

- The test item was checked for possible color interference and possible direct MTT reduction before the study was started.

- Cell viability measurements:
After incubation, the tissues were incubated with 0.3 ml MTT-medium (1.0 mg/ml) for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were transferred to a 6-well plate containing 2 ml isopropanol. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining
cell viability following exposure of the test item.

- Evaluation criteria:
* The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
* The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60%.

- Acceptability criteria:
* The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
* The mean relative tissue viability of the positive control should be <50% relative to the negative control.
* The difference between the % tissue viabilities of the two identically treated replicates should be <20%.
Irritation parameter:
other: mean cell viability (percentage of negative control)
Value:
7.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
29%
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: see 'any other information on results' for historical data. Results for the positive control were within the historical data range and therefore showing that the test system was functioning properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was between >0.8 and <2.5 (i.e., 1.734)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e., 29%)
- The difference between the % tissue viabilities of the two identically treated replicates was <20% (i.e., 3.4, 0.71 and 0.10 for the negative control, the treatment group and the positive control respectively)

- Based on the results, the test substance is considered to be potentially irritating to the eyes and is classified as Category 1 or 2 according to the CLP regulation (Regulation (EC) No. 1272/2008).

Table 1 Historical data

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Positive control

(viability; %)

Range

1.228 – 2.090

0.320 – 0.535

17.80 – 34.18

Mean

1.53

0.42

27.55

SD

0.23

0.07

5.31

n

12

12

12

SD = Standard deviation; n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of January 2013 to May 2017.

Table 2 Individual OD measurements at 570 Nm

 

A

(OD570)

B

(OD570)

Negative control

OD570 measurement 1

OD570 measurement 2

 

1.7689

1.6991

 

1.8253

1.8100

Menadione

OD570 measurement 1

OD570 measurement 2

 

0.1767

0.1732

 

0.1592

0.1561

Positive control

OD570 measurement 1

OD570 measurement 2

 

0.5405

0.5345

 

0.5356

0.5343

OD = Optical density

Duplicate exposures are indicated by A and B.

Interpretation of results:
other: Category 1 or Category 2 according to Regulation (EC) 1272/2008.
Conclusions:
Based on the EpiOcular test, resulting in a mean cell viability of 7.2% compared to the negative control, Menadione is potentially irritant or corrosive to the eyes. The test item should be classified as Category 1 or Category 2 (based on the results no differentiation between categories can be made).
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 June 2017 - 20 June 2017
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
dd. 3 November 2015
Specific details on test material used for the study:
No correction for purity required
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse Vitelco, 's-Hertogenbosch, The Netherlands.
- Storage, temperature and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected an transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.
- Isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum.
Vehicle:
unchanged (no vehicle)
Remarks:
Since no workable suspension of the test substance in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 300.13 - 320.69 (complete coverage of the cornea)

NEGATIVE CONTROL: Physiological saline
- Amount applied: 750 µL

POSITIVE CONTROL: Imidazole 20% (w/w)
- Amount applied: 750 µL
Duration of treatment / exposure:
240 ± 10 minutes
Duration of post- treatment incubation (in vitro):
90 ±5 minutes in sodium-fluorescein for permeability determinations
Number of animals or in vitro replicates:
3 for the negative control, the positive control and the treatment group each.
Details on study design:
TREATMENT METHOD: The medium from the anterior compartment was removed and 750 µL of the negative control and the positive control were introduced onto the epithelium of the cornea. The test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (300.13 to 320.69 mg). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32±1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies).
- POST-EXPOSURE INCUBATION: 90 ±5 minutes in sodium-fluorescein for permeability determinations

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA (see table 1):
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

ACCEPTABILITY CRITERIA:
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
Run / experiment:
Mean of 3 replicates
Value:
13
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
IVIS: 2.7-4.3
Positive controls validity:
valid
Remarks:
IVIS: 131.7-156.4
Other effects / acceptance of results:
- The corneas treated with the test substance showed opacity values between 4.7 and 10.3.
- Permeability values were ranging from 0.018 to 0.861.
- IVIS scores were 7.0, 10.5 and 20.0 (n=3). Since the IVIS scores were > 3 and ≤ 55, no prediction on irritant potency can be made.

OTHER EFFECTS:
- A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed.
- The corneas were turbid yellow after the 240 minutes of treatment with the test substance.
- In the first experiment, one of the negative control eyes was slightly turbid. In the second experiment two of the negative control eye were slightly turbid. This had no effect on the validity of the experiment since the opacity and permeability values were within the historical data range.

ACCEPTANCE OF RESULTS (see table 2 for historical data):
- Acceptance criteria met for negative control: yes, responses for opacity and permeability were less than the upper limits of the historical data range (IVIS ranging from 2.7 to 4.3).
- Acceptance criteria met for positive control: yes, results were within two standard deviations of the historical mean (136.63, SD 27.51 and 2SD 55.02). Corneas were turbid after 240 minutes of treatment.

Table 2 Historical Control Data for the BCOP Studies

 

Negative control

Positive control

 

Opacity

Permeability

In vitro Irritancy Score

In vitro Irritancy Score

Range

-5.4 – 5.2

-0.010 – 0.205

-5.3 – 5.3

86.5 - 211.4

Mean

0.33

0.02

0.53

136.63

SD

1.77

0.02

1.81

27.51

n

110

110

113

111

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2014 to May 2017.

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, no prediction can be made for classification for eye irritation of Menadione since the IVIS was > 3 and ≤ 55 (mean IVIS of 13).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

An in vitro skin irritation study showed that Menadione was irritant to the skin (mean tissue viability of 15%) and should be classified as Category 2 according to GHS and according to Regulation (EC) 1272/2008.

In the in vitro skin corrosion test, Menadione was found to be not corrosive to the skin (mean tissue viability of 100% and 81% after a 3-minute and a 1-hour exposure period, respectively). The test item is not classified according to GHS and according to Regulation (EC) 1272/2008.

Based on the EpiOcular test, resulting in a mean cell viability of 7.2% compared to the negative control, Menadione is potentially irritant or corrosive to the eyes. The test item should be classified as Category 1 or Category 2 (based on the results no differentiation between categories can be made).

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, no prediction can be made for classification for eye irritation of Menadione since the IVIS was > 3 and ≤ 55 (mean IVIS of 13).