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Toxicological information

Skin irritation / corrosion

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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 guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
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.