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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:
weight of evidence
Study period:
20 September 2018 to 31 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Remarks:
The EPISKIN-model is a 3-dimensional human epidermis model obtained after a 13-days culture period of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed human epidermis units are contained in the EpiSkin(TM)SM kits, supplier: SKINETHIC Laboratories; 4, rue Alexander Fleming, 69366 – LYON Cedex 07, France
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN model
- Tissue batch number: 18-EKIN-038

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
The skin units were rinsed thoroughly with approximately 25 mL PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging the epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Linearity range of spectrophotometer: 0.2136 – 3.1752

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is not considered to be corrosive to skin if the mean tissue viability is ≥ 35 % after 4 hours exposure.
- The test substance is considered to be corrosive to skin (Cat 1 A) if the mean tissue viability is < 35 % after 3 min exposure.
- The test substance is considered to be corrosive to skin (Cat 1 B or C) if the mean tissue viability is ≥ 35 % after 3 min exposure and < 35 % after 1 hour exposure OR ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL
NEGATIVE CONTROL
- Amount applied: 50 µL
POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
4 h
Number of replicates:
2

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
103
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
The mean OD value of the two negative control tissues was 0.763.
The positive control result showed 7 % viability.
The difference of viability between the two tissue replicates was between 2.7 % to 9.3 %.
All validity criteria were within acceptable limits. Therefore, the study can be considered as valid.

Any other information on results incl. tables

Table 1: OD values and viability percentages of the controls:

Controls

Optical Density (OD)

Viability (%)

Δ%

Negative Control:
NaCl (9 g/L saline)

1

0.799

105

9.3

2

0.728

95

mean

0.763

100

 

Positive Control:
Glacial acetic acid

1

0.063

8

2.7

2

0.043

6

mean

0.053

7

 

 

Table 2: OD values and viability percentages of the test item (including corrected values):

 

Optical Density (OD)

Viability (%)

Δ%

Test substance

1

0.758

99

7.1

2

0.812

106

mean

0.785

103

 

 

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive, further information necessary to decide on irritation potential and final classification
Conclusions:
The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions.
Executive summary:

The study was conducted to determine the skin corrosion potential of the test item. Disks of EPISKIN (two units) were treated with the test item and incubated for 4 hours (±10 min) at room temperature. Exposure of the test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with 5±1 % CO2 in a ≥ 95% humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 103 %. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential. This result can be used together with further information on the irritative nature of the substance to decide on its final classification.