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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:
15 - 19 Jan 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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted in Jul 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted in Jul 2012
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Mainz, Germany

Test material

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number: 25874
- Production date: 15 Jan 2018
- Delivery date: 16 Jan 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After treatment the tissues were rinsed immediately in 1-minute-intervals.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos Reader 2010 Flexi
- Wavelength: 570 nm
- Filter: no reference filter was used
- Linear OD range of spectrophotometer: 0.1 - 2.5

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by undertaking a MTT cell viability test. The OD (540 - 570 nm) was 1.71 ± 0.09 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 1% Triton X-100. The ET-50 value was determined to be 5.28 h.
- Contamination: The cells for EpiDerm™ tissue were screened for potential biological contaminants, such as HIV-1-virus, Hepatitis B virus, Hepatitis C virus, bacteria, yeast and other fungi. No contamination was detected.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance did not directly reduce MTT.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 µL

NEGATIVE CONTROL
- Amount(s) applied: 30 µL

POSITIVE CONTROL
- Amount(s) applied: 30 µL
Duration of treatment / exposure:
35 min
Duration of post-treatment incubation (if applicable):
23 h and 25 min
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
35 min of exposure with the test item
Value:
90
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The test item was tested for the ability of direct MTT reduction. Since the MTT solution did not change its colour within 1 hour, direct MTT reduction by the test item had not taken place and no data correction was necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD values were 1.677, 1.863 and 1.746 and, thus, in the range of ≥ 0.8 and ≤ 2.8.
- Acceptance criteria met for positive control: The mean viability value was 1.9% and, thus, ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of the three tissues treated with the test substance was 1.1%, treated with the negative control was 5.3% and treated with the positive control was 0.1% and, thus all standard deviations were ≤ 18%.

Any other information on results incl. tables

Table 1: Mean Absorbance Values (OD 570 nm)

Designation Negative Control Reaction mass of potassium sodium phosphotartrate and potassium sodium tartrate and potassium sodium ortho-phosphate (aqueous solution) Positive Control
Mean – blank (tissue 1) 1.677 1.563 0.034
Mean – blank (tissue 2) 1.863 1.592 0.033
Mean – blank (tissue 3)  1.746 1.602 0.036
Mean of the three tissues 1.762 1.586 0.034

Table 2: Tissue Viability (%)

Designation Reaction mass of potassium sodium phosphotartrate and potassium sodium tartrate and potassium sodium ortho-phosphate (aqueous solution) Positive Control
% Tissue viability (tissue 1) 88.7% 1.9%
% Tissue viability (tissue 2) 90.4% 1.9%
% Tissue viability (tissue 3) 90.9% 2.0%
% Tissue viability (mean) 90.0% 1.9%
± SD of mean tissue viability (%) 1.1% 0.1%

Applicant's summary and conclusion

Interpretation of results:
other: no irritating potential according to Regulation (EC) No. 1272/2008
Conclusions:
Under the conditions of the conducted test, the test substance did not show irritating properties in the EpiDerm™ model. The RhE-based test methods are able to identify Cat. 2 and No Cat. chemicals and can thus serve as stand-alone skin irritation methods for non-corrosives.

CLP: not classified