Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-17 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
10 January 2013

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No.: 141767
Purity: 98.1%
Name of test material (as cited in study report): NOPOL
Physical state: colourless to very pale yellow liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 01 April 2015

In vitro test system

Test system:
human skin model
Remarks:
human reconstructed epidermis (tissues)
Source species:
other: human reconstructed epidermis (tissues)
Cell type:
other: human reconstructed epidermis (tissues)
Vehicle:
unchanged (no vehicle)
Details on test system:
Supplier: SkinEthic Laboratories, Lyon, France.
Selection: At receipt, the pH (color of the agar medium) and temperature indicators were checked to ensure the good quality of the tissues before use.
Storage conditions: At receipt, the living EpiskinTM tissues were kept at room temperature in their packaging until required.
Description: The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra-structure and is functionally equivalent to human in vivo epidermis.

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL ± 2 μL of the test item was applied to the epidermis surface.
- Concentration (if solution): undiluted

CONTROLS
- Negative control: Dulbecco’s Phosphate-Buffered Saline (D-PBS)
- Positive control: 5 % (w/v) aqueous solution of Sodium Dodecyl Sulfate (SDS)
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes (± 1 minute).
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 h.
Number of animals:
Triplicate tissues for test item, negative and positive controls
Details on study design:
A new 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.

PRE-INCUBATION OF THE TISSUES ON THEIR DAY OF ARRIVAL (DAY 0)
A volume of 2 mL of pre-warmed maintenance medium was added to the first column of 3 wells of 12-well plates (one plate per item). Then, each EPISKIN™ tissue was transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at 37 °C, 5 % CO2 in a humidified incubator for at least 24 h (± 2 h).

TREATMENT OF TISSUES (DAY 1)
A volume of 2 mL of pre-warmed maintenance medium was pipetted into the second column of 3 wells of the 12-well plates for each item, respectively. Then, each test or control item was applied on three tissues for an exposure period of 15 minutes. The items were applied topically to the corresponding tissues and gently spread over the epidermis surfaces to ensure uniform covering of the tissues. The tissues were processed (treatment and rinsing) in the same order and at regular time-intervals to ensure each tissue received an equal exposure period.
The exposure of the tissues to the test and control items was performed at room temperature for 15 minutes (± 1 minute).

RINSING OF TISSUES AND INCUBATION FOR 42 HOURS (DAY 1)
At the end of the treatment period, each tissue was rinsed with D-PBS to remove any residual test or control items and then incubated for 42 h at 37 °C, 5 % CO2 in a humidified incubator.

MTT VIABILITY ASSAY (DAY 3)
On Day 3, following the 42 h post-exposure incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h (± 5 minutes) at 37 °C, 5 % CO2 in a humidified incubator.

OPTICAL DENSITY MEASUREMENTS (DAY 6)
On Day 6, at the end of the formazan extraction period: The optical density was measured at 570 nm using a plate reader.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
7
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
6
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
7
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
Evaluation of the coloration of tissues at the end of the MTT incubation period: All treated tissues appeared white which was considered to be indicative of dead tissues.
Following a 15 minutes exposure and 42 h of recovery period, the relative mean viability of the tissues treated with the test item was 7 % with a Standard Deviation of 1 % as assessed by the MTT assay. As the mean viability was < 50 % the results met the criteria for an irritant response.

Any other information on results incl. tables

Table 7.3.1/1: Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

Group

Tissue No.

OD measurements

Mean

ODblank

cOD

Mean cOD

Viability (%)

1st

2nd

1st

2nd

 

 

Negative control

1

0.993

1.027

0.037

 

 

0.956

0.990

0.973

101

2

0.975

0.988

0.938

0.951

0.945

98

3

1.019

1.028

0.982

0.991

0.987

102

Positive control

1

0.091

0.090

0.037

 

 

0.054

0.053

0.054

6

2

0.136

0.130

0.099

0.093

0.096

10

3

0.096

0.088

0.059

0.051

0.055

6

Test item

1

0.100

0.105

0.037

 

 

0.063

0.068

0.066

7

2

0.097

0.092

0.060

0.055

0.058

6

3

0.102

0.105

0.065

0.068

0.067

7

OD = optical density

cOD = blank corrected optical density

 

Table 7.3.1/2: Mean tissue viability and Standard Deviations for the test item, the negative and positive controls

 

Group

cOD

Viability (%)

Mean

SD

Mean

SD

Negative control

0.968

0.021

100

2

Positive control

0.068

0.024

7

2

Test item

0.063

0.005

7

1

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Nopol is classified in category 2 (H315) according to Regulation (EC) No. 1272/2008 (CLP) and GHS.
Executive summary:

An in vitro skin irritation study was performed according to Guideline OECD 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.

The test item, Nopol, was applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 h at 37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability). In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.

 

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

 

All treated tissues appeared white, which was considered to be indicative of dead tissues. Following a 15 minute exposure and 42 h of recovery period, the relative mean viability of the tissues treated with the test item was 7% with a Standard Deviation of 1% as assessed by the MTT assay. As the mean viability was < 50% the results met the criteria for an irritant response.

 

Therefore, Nopol is classified in category 2 (H315) according to Regulation (EC) No. 1272/2008 (CLP) and GHS.