Menadione

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

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

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 factor for purity required.

In vitro test system

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

Results and discussion

In vitro

Results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.