[No public or meaningful name is available]

Skin irritation / corrosion

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 guideline

Test guideline 1

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

Test guideline 2

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

Test material information

Constituent 1

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