[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 - 05 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted in 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics

Molecular formula:

not applicable due to UVCB substance

IUPAC Name:

Fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ Human Skin Model

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis
- Tissue batch number: 34102
- Delivery date: 03 November 2020
- Date of initiation of testing: 03 November 2020
- Assay medium lot number: 102920LHB

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing procedure: Rinsing was achieved by filling and emptying each tissue insert under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h (37 °C, 5% CO2)
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 1.704 ± 0.078 (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 5.75 h (acceptance criteria: 4.77-8.72 h).
- Morphology: No histological examination was reported.
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected.
- Reproducibility: No information if the results of the positive and negative controls showed reproducibility over time was reported.

NUMBER OF REPLICATE TISSUES: duplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- The test item did not reduce MTT.

NUMBER OF INDEPENDENT EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 min exposure is less than 50%, or if the viability after 3 min exposure is greater than or equal to 50% and the viability after 1 h exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 min exposure is greater than or equal to 50% and the viability after 1 h exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Lot/batch no.: 18L10BA1A
- Purity: 100%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration: 8N
- Lot/batch no.: H3410

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

Duplicates for each treatment and control group (3 and 60 min).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

91.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

85.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.
- Colour interference with MTT: The test substance did not change the colour, when mixed with deionised water and thus passed the colour interference pre-test. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.862 and 2.233).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 h was < 15% compared to the negative control (3.7%).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (values between 1.1% and 12.8%)

Any other information on results incl. tables

Table 2: Results (summary)

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.9	91.7
60 minute	100*	3.7	85.7

*: The mean viability of the negative control tissues is set at 100%

Table 3: Detailed results

Tissue	Exposure Period	MeanOD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	2.233	2.048	0.262	12.8	100*
		1.862
	60 Minutes	2.006	2.022	0.023	1.1
		2.038
Positive Control	3 Minutes	0.095	0.080	0.022	na	3.9
		0.064
	60 Minutes	0.096	0.076	0.029	na	3.7
		0.055
Test Item	3 Minutes	1.953	1.879	0.105	5.6	91.7
		1.805
	60 Minutes	1.755	1.733	0.032	1.8	85.7
		1.710

OD: Optical density

*: The mean percentage viability of the negative control tissue is set at 100%

na: Not applicable

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat. 1, 1A, 1B/C) based on a positive result in the Reconstructed human epidermis test method (in vitro skin corrosion). A negative in vitro corrosivity response is not conclusive with respect to non-classification or classification as a skin irritant and therefore requires further evaluation and/or data generation.