Menadione

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Menadione

EC Number:

200-372-6

EC Name:

Menadione

Cas Number:

58-27-5

Molecular formula:

C11H8O2

IUPAC Name:

2-methyl-1,4-dihydronaphthalene-1,4-dione

Test material form:

solid: particulate/powder

Details on test material:

- Physical appearance: yellow crystalline powder
- Storage conditions: at room temperature protected from light

Specific details on test material used for the study:

No correction for purity required

Test animals / tissue source

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).

Test system

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%.

Results and discussion

In vitro

Results

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).

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).