Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Mar - 30 Mar 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:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek Corporation, Ashland MA, USA
Source strain:
other: EpiDerm Skin Model (EPI-200)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200) (MatTek Corporation, Ashland MA, USA)
- Tissue batch number: 28307, kit E and F

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 ± 0.5 min exposure); 37.0 ± 1.0 °C (60 ± 5 min exposure)
- Temperature of post-treatment incubation: 37.0 ± 1.0 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed with phosphate buffered saline to remove residual test item; not further specified
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (1 mg/mL)
- Incubation time: 3 h
- 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 undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.851 ± 0.085 (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 7.24 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. None of these were detected.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable as the test item did not show reducing capacity 1 h after MTT incubation.

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

PREDICTION MODEL / DECISION CRITERIA
Step 1
- The test item is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% and the viability after 1 hour exposure is less than 15%.
- The test item is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Step 2 (for test items identified as corrosive in Step 1)
- Sub-category 1A: if the viability after 3 minutes exposure is less than 25%
- A combination of sub-categories 1B and 1C : if the viability after 3 minutes exposure is greater than or equal to 25%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
- Concentration : 8 N

NEGATIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
3 min and 60 ± 5 min
Number of replicates:
duplicates for each treatment and control group

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean values of 2 tissues
Run / experiment:
3 min exposure
Value:
97
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean values of 2 tissues
Run / experiment:
60 min exposure
Value:
25
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 1 h incubation with MTT
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water.

ACCEPTANCE OF RESULTS:
- The mean OD of the tissue replicates treated with the negative control are within the laboratory historical control data range, i.e. ≥ 1.258.and ≤ 2.615 for every exposure time (range: 1.871 to 2.120).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (6.2%).
- Acceptance criteria met for variability between replicate measurements: The Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 2.7% to 15%).

Any other information on results incl. tables

Table 2. Mean absorption values

 

3-minute application

1-hour application

A (OD570)

B (OD570)

Mean  ±

(OD570)

SD

A (OD570)

B (OD570)

Mean ±

(OD570)

SD

Negative control

1.871

1.923

1.897

±

0.037

2.120

2.112

2.116

±

0.006

N-ethylcaprolactam

1.918

1.751

1.834

±

0.118

0.532

0.518

0.525

±

0.010

Positive control

0.212

0.244

0.228

±

0.023

0.143

0.122

0.132

±

0.015

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0414). Isopropanol was used to measure the background absorption.

Table 3. Mean tissue viability

 

3-minute application

viability

(percentage of control)

1-hour application

viability

(percentage of control)

Negative control

100

100

N-ethylcaprolactam

97

25

Positive control

12

6.2

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive according to Regulation (EC) No. 1272/2008
Conclusions:
Applying a worst case approach, the test substance is classified as a Skin Irrit. 2, H315 according to Regulation (EC) No. 1272/2008 until an in vitro skin irritation study is conducted to determine whether or not the test item is a skin irritant.
CLP: Skin Irrit. 2, H315 according to Regulation (EC) No. 1272/2008