Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 20, 2017 to May 13, 2017
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Optical Density (OD) values obtained with blanks were higher than 0.1 (0.184) causing a deviation from Acceptance Criteria. However, this is not considered to be an issue in the interpretation of this study data.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Rape oil, reaction products with diethylenetriamine, di-Me sulfate-quaternized
EC Number:
308-732-5
EC Name:
Rape oil, reaction products with diethylenetriamine, di-Me sulfate-quaternized
Cas Number:
98219-63-7
Molecular formula:
C50H97N3O5S1 (Fatty amides C44:2- representative)
IUPAC Name:
Rape oil, reaction products with diethylenetriamine, di-Me sulfate-quaternized
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed human epidermal model EpiDermTM
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:
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.

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:
Single topical application of 25 mg of neat test substance.
Duration of treatment / exposure:
60 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, 95% RH).
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Three tissues per condition (n=3).

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
96.6
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
All validity criteria for the test were met:
- Criteria: the mean OD570 of the negative control (treated with DPBS) tissues is ≥ 0.8 and ≤ 2.8.
Result for the test: 1.445

- The mean of the positive control relative percentage viability must be ≤ 20 % of the mean of the negative controls.
Result for the test: 2.7 %

- The standard deviation of OD values for triplicate skin models in each expeimental condition must be < 18 %.
Results for the test:
NC: 6.02 %
PC: 1.11 %
Test substance: 1.62 %

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.184) 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 XCellR8 laboratory and meet our current internal acceptance criteria of blank OD values <0.194 (mean of XCellR8 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
Conclusions:
Under the study conditions, the percentage viability for the test substance was established at 96.6% in EpiDerm™ model, therefore the test substance is considered to be non-Irritating to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, ‘di-C16-18 -satd. and C18-24-unsatd. AAEMIM-MS (active: 100%)' using Reconstructed Human Epidermis (RHE) cells, according to OECD 439 Guideline, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, 95% RH. On Day 1, the tissues in triplicate were exposed to nominal 25 mg of test substance and 30 µL reference substances, applied topically for 60 ±1 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, 95% RH), followed by rinsing steps and a 42 ± 4 h post-dose incubation at 37°C, 5% CO2, 95%RH). On Day 2, the medium was changed and on Day 3, MTT viability test with readings at 570 nm without reference filter was performed. 30 µL of DPBS and 5% SDS were used as negative control and positive control, respectively. 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 96.6%, 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 be non-irritating to the skin (XCellR8, 2017).