Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 16th to December 1st, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Remarks:
SkinEthic™ RHE model
Source species:
human
Cell type:
other: normal human keratinocytes.
Cell source:
other: human adult donor, not specified.
Source strain:
not specified
Justification for test system used:
The SkinEthic™ RHE model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 16 MPE 128
- Delivery date: 29/11/2016
- Expiration date: 05/12/2016
- Date of initiation of testing: 16/11/2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.3 (CV 1.3%)
- Barrier function: 4.3 h
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
41 hours and 12 minutes.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
66.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
(DPBS)
Positive controls valid:
yes
Remarks:
1.0% viability (5% SDS)
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean corrected percent viability of the treated tissues was 66.7 %, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The standard deviation of the negative control group was 21.7%, instead of ≤ 18% as initially scheduled. However, considering the results obtained, this deviation is considered as without impact on the conclusion of the study.
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1. Summary of results.

 

Skin

OD

Mean OD /disc (#)

Mean OD / product

Viability

%

Mean viability

%

SD

Viability

Conclusion

Negative

Control

1

1.541

1.619

1.316

123.1

100.0

21.7

 

1.650

1.668

2

1.268

1.276

97.0

1.271

1.290

3

1.032

1.052

80.0

1.044

1.082

Positive

Control

4

0.013

0.014

0.013

1.1

1.0

0.1

Irritant

0.016

0.013

5

0.015

0.013

1.0

0.013

0.013

6

0.012

0.011

0.8

0.011

0.011

Test item

21

0.814

0.856

0.877

65.1

66.7

2.0

Non-irritant

0.878

0.877

22

0.868

0.869

66.1

0.878

0.862

23

0.897

0.907

68.9

0.905

0.921

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria (not irritant).
Conclusions:
The mean corrected percent viability of the treated tissues was 66.7 %, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.
Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic™ RHE epidermis model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 41 hours 12 minutes in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Under test conditions, the mean corrected percent viability of the treated tissues was 66.7 %, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.