[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:

14 - 18 Dec 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2019

Deviations:

yes

Remarks:

demonstration of technical proficiency not included in the study report

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate

Cas Number:

18641-58-2

Molecular formula:

C69H132O8

IUPAC Name:

2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek Corporation (Ashland, Massuchusetts, USA)

Source strain:

other: EpiDerm™ Skin Model (EPI-200)

Vehicle:

unchanged (no vehicle)

Remarks:

The skin was moistened with 25 µL Milli-Q-water to ensure close contact of the test item to the tissue.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Ashland, Massachusetts, USA)
- Tissue batch number: 33783 Kit F
- Date of initiation of testing: 14 Dec 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature and 60 min at 37 ± 1.0 °C (actual range: 35.9 – 37.3 °C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL, 300 µL/well
- Incubation time: 3 h at 37 ± 1.0 °C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate 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 1.9 ± 0.157 (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 5.81 h (acceptance criteria: 4.77-8.72 h).
- 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.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable as the test item did not show colour changing or reducing capacity after incubation with MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Single experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered to be corrosive to skin if the viability after 3 min exposure is less than 50% and the viability after 1 h exposure is less than 15%.
- The test item 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: 25.14 - 44.96 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8.0 N

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

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The test item did not show reducing capacity after incubation with MTT
- Colour interference with MTT: The test substance did not change colour in the presence of MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not included by the testing laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD 570 nm of the tissue replicates treated with the negative control was 1.780 ± 0.006 for the 3 min exposure and 1.732 ± 0.186 for the 1 h exposure and fell within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was < 15% compared to the negative control (6.8% for 3 min exposure, 7.2% for 1 h exposure).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) in the range 20 – 100% viability between tissue replicates was ≤ 30% (values between 0.4 - 27%).

Any other information on results incl. tables

Table 1: Mean absorption in the in vitro skin corrosion test (values were corrected for background absorption)

Substance	3-min application	1-h application
	Mean ± SD (OD570)	Mean ± SD (OD570)
Negative control	1.780 ± 0.006	1.732 ± 0.186
Test item	1.668 ± 0.140	1.763 ± 0.238
Positive control	0.120 ± 0.013	0.125 ± 0.027
SD = Standard deviation

Table 2: Mean tissue viability in the in vitro skin corrosion test

Substance	3-min application	1-h application
	viability (percentage of control)	viability (percentage of control)
Negative control	100	100
Test item	94	102
Positive control	6.8	7.2

Table 3: Historical control data^#generated in the testing facility

	Negative control		Positive control
	3-min treatment (OD570)	1-h treatment (OD570)	3-min treatment (OD570)	1-h treatment (OD570)
Range	1.375 - 2.226	1.317 - 2.269	0.080 - 0.671	0.032 - 0.319
Mean	1.752	1.769	0.173	0.135
SD	0.175	0.170	0.086	0.048
n	122	122	122	122
SD = Standard deviation
n = Number of observations

#: Data were collected over the period Nov 2017 – Nov 2020

 

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive according to Regulation (EC) No. 1272/2008.

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 human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.