Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2018 to January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Density: 0.8-1.00 g/cm3
- Moisture content : less than 10%
- Residual solvent:-
- Stabilisation: stable
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Oxalate de Sodium
- Batch No.of test material: Batch No 1800703
- Expiration date of the batch: 22 May 2023
- Purity: 99.2%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: opaque plastic flask, at ambient temperature
- Stability under test conditions: Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied, as supplied.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Foreskin
Justification for test system used:
Test system recommended in the OECD 439 Guideline.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin SA, RHE/S/17 (SkinEthic RHE model)
- Tissue batch number: Batch No. 18-RHE-132
- Delivery date: received on 06 November 2018
- Date of initiation of testing: 06 November 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The test item was applied at room temperature
- Temperature of post-treatment incubation: incubation at 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 42 minutes after the test item application, the nylon mesh was removed and the human epidermises were washed with 25 x 1 mL of DPBS.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: The skin samples were placed in 300 μL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2.
- Incubation time: 41 hours and 20 minutes post-treatment incubation period in fresh medium at 37°C, 5% CO2.
- Spectrophotometer: The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The OD values obtained for each test item are used to calculate a percentage of viability relative to the negative control, which is arbitrarily set to 100%.
The results were expressed as a viability percentage compared with the negative control: Viability % = (OD test item / OD negative control) x 100

NUMBER OF REPLICATE TISSUES: Triplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
There is no direct interaction between the test item and MTT.

PREDICTION MODEL / DECISION CRITERIA
The test item is considered as non-irritant to skin in accordance with UN GHS No Category if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
The test item is identified as requiring classification and labelling according to UN GHS (Category 2) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non-corrosive”.
The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: dose of 16 mg

VEHICLE
Applied as supplied

NEGATIVE CONTROL
- Amount applied: 16 μL of distilled water – ADL Prochilab - Batch No. 180517

POSITIVE CONTROL
- Amount applied: 16 μL of 5% sodium dodecyl sulfate (SDS)
- Concentration: 5 % SDS
Duration of treatment / exposure:
42 minutes at room temperature
Duration of post-treatment incubation (if applicable):
41 hours and 20 minutes post-treatment incubation period in fresh medium at 37°C, 5% CO2.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
105.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

 

Well ID

OD

Mean OD / disc

Mean OD / product

Viability %

Mean viability %

SD viability

Conclusion

Negative control

SPL 1

0.961

1.017

0.948

0.975

0.884

110.3

100.0

9.2

 

SPL 2

0.851

0.858

0.871

0.860

97.2

SPL 3

0.809

0.817

0.830

0.818

92.5

Positive control

SPL 4

0.009

0.010

0.010

0.009

0.010

1.0

1.1

0.1

Irritant

SPL 5

0.012

0.011

0.011

0.011

1.2

SPL 6

0.010

0.011

0.011

0.010

1.1

Test item PH-18/0521

SPL 16

0.889

0.987

0.986

0.954

0.934

107.9

105.7

3.4

Non irritant

SPL 17

0.950

0.945

0.952

0.949

107.3

SPL 18

0.882

0.891

0.929

0.900

101.8

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In accordance with the Regulation EC No. 1272/2008, the test item Oxalate de Sodium has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.
No hazard statement or signal word is required.
Executive summary:

The aim was to evaluate the possible irritating effects of the test item Oxalate de sodium after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 439.

The test item Oxalate de Sodium was applied as supplied, during 42 minutes, at the dose of 16 mg to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) previously moistened with 10 μL of distilled water. The application was followed by a rinse with 25 mL of DPBS and a 41 hours and 20 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The mean percent viability of the treated tissues was 105.7% (considered as 100%), versus 1.1 % in the positive control (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation EC No. 1272/2008, the test item Oxalate de Sodium has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.

No hazard statement or signal word is required.