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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:
2018-06-14 to 2018-09-06
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.40bis (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: no data
Justification for test system used:
As recommended by OECD guideline
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number(s): EPI-200-SCT, Lot no. 28629
- Date of initiation of testing: 2018-06-19

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Not specified
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one washing step, volume not specified
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: Tecan Sunrise Magellan Version 6.4
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540-570 nm) [1.0 -3.0]: Result: 1.884 ± 0.1525
- Barrier function: ET-50 assay, 100 µL 1% Triton X-100, 4 time-points, n = 3, MTT assay, ET-50 [4.77-8.72 h]: Result: 4.79 h
- Contamination: Longe term antibiotic and antimycotic free culture, No contamination: Result: Sterile

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- N. of replicates : 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg moistened with 25 µL deionized water

VEHICLE
- Amount(s) applied (volume or weight with unit): Deionized water 25 µL
- Lot/batch no. (if required): 180958001

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL deionized water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8 N KOH
Duration of treatment / exposure:
1st period: 3 min
2nd period: 60 min
Duration of post-treatment incubation (if applicable):
3 h incubation with MTT
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
3-minutes exposure
Run / experiment:
tissue 1
Value:
103.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
3-minutes exposure
Run / experiment:
tissue 2
Value:
99.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
60-minutes exposure
Run / experiment:
tissue 1
Value:
103.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
60-minutes exposure
Run / experiment:
tissue 2
Value:
100.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Colour interference with MTT: No


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline:

Short time incubation – 3 min
Negative control: Mean OD 1.624 (2.5±2.4) Average viability [%] 100 (2.5 ± 2.4), Viability range [%]: 93.7–106.3; % difference 0.14–8.6, unqualified experiments: 0#1
8 N KOH: Mean OD0.126 (8.8±7.4) Average viability [%] 7.6 (8.8 ± 7.4), Viability range [%]: 2.0–15.6; % difference <0.01–23.7, unqualified experiments: 0
Long time incubation – 60 min
Negative control
Mean OD 1.650 (4.0±5.0), Average viability [%] 100 (4.0±5.0), Viability range [%]: 85.6–114.4; % difference 0.13–18.3, unqualified experiments: 0#1
8 N KOH
Mean OD 0.090 (5.7±9.8), Average viability [%] 5.9, Viability range [%]: (5.7 ± 9.8); % difference 2.0–12.6 0.30–38.4, unqualified experiments: 0#2
#1 Unqualified results = if the mean OD of the NC tissues is < 0.8 or > 2.8 if difference in viability for duplicate tissues > 30%
#2 Unqualified results = 8 N KOH: viability > 15% (1-h exposure)

Any other information on results incl. tables

Table 1: Viabilities after both exposure durations

 

Viability 3-minutes exposure

Viability 60-minutes exposure

 

Optical density [OD540] mean tissue 1 and 2 (percent differences)

Mean viability (Difference of Viability) [%]

Optical density [OD540] mean tissue 1 and 2 (percent differences)

Mean viability (Difference of Viability) [%]

Negative control (deionized water)

1.564

100.0

1.606

100.0

 

(2.8)

(2.7)

(0.6)

(0.5)

TA-3

1.584

101.3

1.639

102.0

 

(3.7)

(3.7)

(2.5)

(2.6)

Positive control (8 N KOH)

0.068

4.4

0.070

4.4

 

(11.8)

(0.0)

(0.0)

(0.0)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions of the study, the test item tested at two exposure periods of 3 minutes or 1 hour was non-cytotoxic and, hence, predicted to be non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin. Therefore, the test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not corrosive to the skin.