Registration Dossier - ECHA (2024)

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 - 22 Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Materials and methods

Test guidelineopen allclose all

GLP compliance:

yes (incl. QA statement)

Test material

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small model (EPISKIN-SM™)

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™ (SkinEthic Laboratories, Lyon, France)
- Tissue batch number: 21-EKIN-046
- Production date: 16 Nov 2021
- Date of initiation of testing: on the day of tissue receipt

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature
- Temperature of post-treatment incubation: 37.0 ± 1.0°C (actual range 36.4 - 36.9°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 h ± 5 min at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: The IC50 determined by MTT test after incubation with SDS was 1.8 mg/mL (acceptance criteria: 1.5 mg/L ≤ IC50 ≤ 3.0 mg/L).
- Morphology: Multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum. The number of cell layers was 8.
- Contamination: On cells from the donors, the absence of bacteria, fungus and mycoplasma was verified. On blood of the donors, the absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs were verified.

TESTING OF INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
- COLOR INTERFERENCE: The test item was checked for possible color interference before the study was started. 50 µL of the test item or 50 µL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 h at 37.0 ± 1.0°C in the dark. Furthermore, 50 µL of the test item or 50 µL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 h at room temperature with gentle shaking. At the end of the exposure time, the mixtures were centrifuged for 30 sec at 16000 g if needed and the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.

- MTT REDUCTION: The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µL of the test item was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 h at 37.0 ± 1.0°C in the dark. A negative control, 50 µL sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant (Cat. 2) or corrosive (Cat. 1) to skin if the relative mean viability of 3 individual tissues after 15 min of exposure and 42 h of post incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant (no category) to skin if the relative mean viability of 3 individual tissues after 15 min of exposure and 42 h of post incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 µL
- Concentration: neat test material

NEGATIVE CONTROL
- Amount applied: 25 µL

POSITIVE CONTROL
- Amount applied: 25 µL
- Concentration: 5%
- The positive control was re-spread after 7 min contact time.

Duration of treatment / exposure:

15 ± 0.5 min at room temperature

Duration of post-treatment incubation (if applicable):

42 ± 1 h at 37°C

Number of replicates:

triplicates for each treatment and control group

Results and discussion

In vitro

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Because no color change was observed in the presence of MTT it was concluded that the test item did not interact with the MTT endpoint. An additional functional check with freeze-killed or viable tissues was not performed.
- Color interference with MTT: Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0003 and 0.0011, respectively. Therefore, it was concluded that the test item did not induce color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability of 2.5% after 15 ± 0.5 min exposure.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was ≤ 16%.

Any other information on results incl. tables

Table 1:Mean Absorption in the In Vitro Skin Irritation Test

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.046	1.181	1.243	1.157	±	0.101
Test item	1.127	1.351	1.483	1.320	±	0.180
Positive control	0.026	0.031	0.029	0.028	±	0.003

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.044). Isopropanol was used to measure the background absorption.

Table 2:Mean Tissue Viability in the In Vitro Skin Irritation Test

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	8.7
Test item	114	16
Positive control	2.5	0.2

Table 3:Historical Control Data for In Vitro Skin Irritation Studies

	Negative control (absorption; OD570)	Positive control (absorption; OD570)
Range	0.507 - 1.478	0.026 - 0.549
Mean	1.060	0.106
SD	0.153	0.085
n	159	159

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2018 to May 2021.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

In the EPISKIN test according to OECD TG 439 the relative mean tissue viability obtained after 15 ± 0.5 min treatment with the test item compared to the negative control tissues was 114%. Since the mean relative tissue viability for the test item was above 50%, the test item is considered to be non-irritant.