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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 February 2017 - 17 February 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:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
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 material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Name as cited in report: X-19924
Appearance: thick amber paste
Storage: at room temperature
Specific details on test material used for the study:
Purity correction factor: 1.14
Stability at higher temperatures: stable at a maximum temperature of 60°C for a maximum duration of 30 minutes

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
Details on animal used as source of test system:
EpiDerm™ Reconstructed Human Epidermis (Lot no.: 24982 kit H and I)
- All cells used to produce Eipderm™ are purchased or derived from tissue obtained by MatTek Corporation from acredited institutions.
- Cells are screened for potential biological contaminants (HIV-1, Hepatitis B, Hepatitis C, bacteria, yeast and fungi)
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)
- Tissue lot number: 24982 kit H and I
- Surface: 0.6 cm^2
- The model consists of keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment at 37.0 ± 1.0°C (actual range: 36.6 - 37.0°C)

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

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

NUMBER OF REPLICATE TISSUES: 2 replicates per exposure duration (4 replicates per test item), one negative control, one positive contol.

ACCEPTANCE OF RESULTS:
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 acceptance limits of OECD 431 (lower limite ≥0.8 and upper limit ≤2.8)
b) The mean relative tissue viability following 1-hour exposue to the positive control should be <15%
c) In the range 20-100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤30%.

DECISION CRITERIA: see table 1
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
other: concurrent control for MTT reduction by test item
Amount/concentration applied:
Excess amount of the paste, spread directly on top of skin moistened with 25 µL Milli-Q water
Duration of treatment / exposure:
3-minute and 1-hour
Duration of post-treatment incubation (if applicable):
none

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
107
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(100%)
Positive controls validity:
valid
Remarks:
(29%)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
90
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(100%)
Positive controls validity:
valid
Remarks:
(9%)
Other effects / acceptance of results:
- Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered to be non corrosive.

- OTHER EFFECTS:
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes, mean relative tissue viability following 1-hour exposure was 9%.
- In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤12% for the test item and negative control. For the positive control, the Coefficient of Variation between tissue replicates at the 3-minute exposure was 39%. Since both individual tissues of the positive control were clearly positive it was concluded that the test system functioned properly.

Table 2 shows the results of individual OD measurements at 570 nm.

Any other information on results incl. tables

Table 2 Individual OD measurements at 570 nm.

 

3-minute application (OD570)

1-hour application (OD570)

 

A

B

A

B

Negative control

Measurement 1

Measurement 2

Measurement 3

1.4908

1.4052

1.4427

1.4308

1.3742

1.4020

 

1.7312

1.7305

1.6864

1.5932

1.5681

1.5657

X-19924

Measurement 1

Measurement 2

Measurement 3

 

1.6368

1.6057

1.5798

 

1.4898

1.3770

1.4145

 

1.4855

1.4863

1.4874

 

1.4870

1.4816

1.5166

Positive control

Measurement 1

Measurement 2

Measurement 3

 

0.5413

0.5453

0.5385

 

0.3585

0.3446

0.3422

 

0.1999

0.2067

0.2032

 

0.1843

0.1793

0.1789

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
An in vitro skin corrosion test was conducted with X-19924 according to OECD 431 guideline and GLP principles. It is concluded that this test is valid and that X-19924 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

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