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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 28, 2017 to September 01, 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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
However, Guideline or SOP 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: paste

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Source strain:
not specified
Details on animal used as source of 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.
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:
water
Details on test system:
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# 25839) were checked in-house for MatTek acceptance ranges with 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) test substance (62.8% active)
Duration of treatment / exposure:
60 ± 1 minute (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, ≥95% RH)
Duration of post-treatment incubation (if applicable):
42 ± 4h
Number of replicates:
Three tissue replicates per condition

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
and c(Compared to the negative control)
Run / experiment:
Mean of 3 replicates
Value:
ca. 5.741
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

Results

Prior to the study, the required compatibility checks confirmed that the test substance did not interfere with MTT. However, water colouration was observed. Therefore, additional viable tissues were run without MTT to control for colouration (Colourant Controls/No MTT). The background levels of interference from this control was subtracted from the initial viability derived from the main test to give an adjusted overall viability.

 

Table 1: Results summary

 

Percentage of viability

(relative to negative control)

Classification

Irritant (I)/non irritant (NI)

Test substance

5.741%

Irritant (I)

The test substance reduced the viability to below 50% and should be considered as Irritant to the skin.

Note that the final value was corrected after assessment of the colourant control.

 

Data Analysis:

 

Table 2: Viability measurements after 60 min (± 1min) of application and 46h (+9 mins) post-incubation of test and reference substances and controls

Condition

Tissue #

Raw data

Blank corrected data

Mean OD

% of Viability

Aliquot 1

Aliquot 2

Aliquot 1

Aliquot 2

NC

Tissue 1

1.911

1.865

1.737

1.691

1.714

105.0

Tissue 2

1.265

1.371

1.091

1.197

1.144

77.8

Tissue 3

1.856

1.595

1.682

1.421

1.552

95.0

PC

Tissue 1

0.265

0.282

0.091

0.108

0.100

6.1

Tissue 2

0.232

0.232

0.058

0.058

0.058

3.6

Tissue 3

0.231

0.291

0.057

0.117

0.087

5.3

TA3

Tissue 1

0.304

0.303

0.130

0.129

0.130

8.0

Tissue 2

0.385

0.376

0.211

0.202

0.207

12.7

Tissue 3

0.286

0.288

0.112

0.114

0.113

6.9

NC: negative control (DPBS), PC: Positive control (SDS 5%), TA3: Test substance. 

Note: Rounded figures used. NC tissue 2 was removed from further analysis as it was an outlier value as can be seen from the OD values and % viability.

 

Table 3: Mean and SD of cell viability measurements and of viability percentages after 60 min (± 1min) of application and 46h (+9 mins) post-incubation

Name

Code

Mean of OD

SD of OD

Mean of viability (%)

SD of viability (%)

CV %

Classification

DPBS

NC

1.633

0.115

100

7.036

7.036

Non-Irritant

SDS 5%

PC

0.082

0.021

5.011

1.304

26.016

Irritant

Test substance

TA3

0.150

0.050

9.185

3.056

33.270

Irritant

NC: Negative control (DPBS), PC: Positive control (SDS 5%), TA3: Test substance.

Prediction model of irritancy: test substances that reduce the viability to 50% or below are irritant (I), test substances with a percentage viability above 50% are considered to be non-irritant (NI).

Note: Rounded figures used.

Table 4: Mean % viabilities obtained from additional tests to correct for colourant interference

Test Substance

Colourant Controls (CC/No MTT): % viability

Initial % Viability

Corrected % Viability

TA3

3.444

9.185

5.741%

 TA3: Test substance.

Evaluation of the results

Results were checked against the following acceptance criteria: 

 

Description

Actual values

PASS/FAIL

Acceptance criterion 1

The mean OD570of the

negative control (treated with DPBS) tissues is≥ 0.8 and ≤ 2.8

1.633

PASS

Acceptance criterion 2

The mean of the positive control relative percentage viability must be ≤ 20% of the mean of the negative controls

5.011

PASS

Acceptance criterion 3

The standard deviation of OD values for triplicate skin models in each experimental condition must be < 18%

NC: 7.036

PC: 1.304

TA3: 3.056

PASS

Acceptance criterion 4

The mean OD of the 6 wells containing extraction solvent alone (blanks) should be ≤ 0.1

0.174

FAIL

All acceptance criteria were met with the exception of criterion 4:

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) 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 laboratory internal acceptance criteria of blank OD values <0.194 (mean of XCellR8 historical data, based on blanks obtained during 66 historical runs), therefore this is not considered to be an issue in the interpretation of this study data. The study author concluded that this SOP and guideline deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained.

 

Interpretation of results following Prediction Model

 

1) A test substance is considered to be an irritant (I) to skin in accordance with UN GHS Category 2 or EU R38 if the skin model viability after exposure and post-treatment incubation is ≤50%.

 2) A test substance may be considered as a non-irritant (NI) if the skin model viability after exposure and post-treatment incubation is >50%.

The final percentage of viability obtained with the test substance test substance was 5.741%, therefore it was considered as Irritant to the skin.

Applicant's summary and conclusion

Interpretation of results:
other: Category 2 (irritant) based on EU CLP criteria
Conclusions:
Under study conditions, the test substance was determined to be irritating to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool' (active: 62.8%) using Reconstructed Human Epidermis (RHE) test method, according to OECD Test Guideline 439, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% relative humidity (RH), after which 30 µL test substance and reference substances (negative control: sterile Dulbecco’s phosphate buffered saline and positive control: sodium dodecyl sulphate (5% in water)) were applied topically for 60 ± 1 minute in triplicate, followed by rinsing steps and a 46 h + 9 mins post-dose incubation at 37°C, 5% CO2, ≥95% RH. Medium was changed on Day 2. MTT viability test was conducted and readings at 570 nm without reference filter were taken on Day 3. A test substance is considered to be an irritant to skin if the skin model viability after exposure and post-treatment incubation is ≤50%. Based on the results, the final percentage of viability obtained with the test substance was determined to be 5.741%; therefore it was considered as irritating to the skin. The Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) causing a deviation from Acceptance Criteria 4. The study author concluded that this deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained. Hence, the study results qualified the acceptance criteria. Under the study conditions, the test substance was determined to be irritating to the skin (XCellR8, 2017).