Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 05, 2018 to April xx, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Principles of method if other than guideline:
Optical Density (OD) values obtained with blanks were higher than 0.1 (0.191) causing a deviation from Acceptance Criteria 4. However, this SOP and guideline deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: White pastilles

In vitro test system

Test system:
human skin model
Remarks:
MatTek EpiDermTM tissue model EPI-200
Justification for test system used:
Initially the predictive capacity of the modified EpiDerm™ Skin Irritation Test (SIT) test method, using MatTek EpiDermTM tissue model EPI-200, underwent full prospective validation from 2003-2007. The test method components of this method were used to define the essential test methods components of the original and updated ECVAM Performance Standards (PS). A modification of the original EpiDerm™ SIT was validated using the original ECVAM PS in 2008. In 2008, ESAC concluded that the Modified EpiDerm™ SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances.
Vehicle:
unchanged (no vehicle)
Details on test system:
Test system:
The reconstructed human epidermal model EpiDermTM (EPI-200-MatTek Corporation) consists of normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Characterisation of the test system:
MatTek’s EpiDermTM model has been extensively characterised for multiple parameters including morphology, tissue viability, skin barrier function and sterility. QC results for the specific lot of models received (Lot# 25819) were checked in-house for MatTek acceptance ranges with the following outcome:

- Morphology - PASS
- Tissue viability - PASS
- Skin barrier function (ET50 value for 1 % Triton X-100) where ET50 is the time taken for 1 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control) - PASS
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg (nominal) of the neat test substance
Duration of treatment / exposure:
60 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5 % CO2, >=95% relative humidity).
Duration of post-treatment incubation (if applicable):
42 h post-treatment incubation.
Number of replicates:
3 replicates for test substance, negative and positive control.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
103.13
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- Prior to the study, the required compatibility checks (as per SOP L0029) confirmed that the test substance did not interfere with MTT and no water colouration was observed.
- The test substance did not reduce the viability below 50% and therefore should be considered as non-irritant to the skin.

All acceptance criteria were met with the exception of 1 criterion:

- The mean OD570 of the negative control (treated with DPBS) tissues is ≥0.8 and ≤2.8.
Result: 1.777
- The mean of the positive control relative percentage viability must be ≤20 % of the mean of the negative controls.
Result: 3.8 %
- The standard deviation of OD values for triplicate skin models in each experimental condition must be <18 %.
Results:
NC: 5 %
PC: 0.72 %
Test substance: 16.17 %
- The mean OD of the 6 wells containing extraction solvent alone (blanks) should be ≤0.1.
Result: 0.191

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.191) causing a deviation from Acceptance Criteria 4. However, the spectrophotometer was fully validated and had passed all required tests. The OD values for blanks observed in this study are consistent with historical data using this spectrophotometer in the laboratory and meet our current internal acceptance criteria of blank OD values <0.194 (mean of historical data, based on blanks obtained during the last 66 studies), therefore this is not considered to be an issue in the interpretation of this study data.
This SOP and guideline deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained.

Applicant's summary and conclusion

Interpretation of results:
other: CLP criteria not met
Remarks:
non-irritant to skin
Conclusions:
Under the study conditions, the test substance was determined to be non-irritant to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, ' Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide' (UVCB), in Reconstructed Human Epidermis (RHE) cells, according to OECD Guideline 439, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative control for 60 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5 % CO2, 95% RH) and 42 h post incubation period. Test was performed with 3 replicates for each type of treatment. Tissues were first pre-wetted with 25 μL DPBS (Sterile Dulbecco’s Phosphate Buffered Saline), subsequently 25 mg (nominal) of the neat test substance was applied. 30 μL of DPBS was used as negative control and 5% of sodium dodecyl sulphate as positive control. Viability of the tissues was assessed in MTT test and compared to the negative control. The percentage of viability obtained with the test substance was 103.13%, which is well above the irritant limit of 50%. The study met all the validity criteria. Under the study conditions, the test substance was determined to be non-irritant to the skin (XCellR8, 2017).