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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:
19. - 21.April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant

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 (incl. certificate)
Remarks:
Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2017/33, dated 27 April 2017

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Lot/batch No.of test material: 05297/MA
- Expiration date of the lot/batch: Retest date October 2021
- Storage condition of test material: room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed epidermis of normal human keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
Human skin model
The 0.50 cm² reconstructed epidermises (Episkin SA, RHE/S/17 Batch No. 19-RHE-041) were received on 19 March 2019. The same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 300 µL of growth medium (Episkin SA, batch No. 19 SGM 031) during 2 hours and 20 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA, batch No. 19 SMM 011).

Technical Data, safety sheet and certificate of analysis:
SkinEthic RHE/Human Epidermis (RHE/S/17): 0.5 cm² reconstructed epidermis for normal human keratinocytes. Cells are grown on inert polycarbonate filters in chemically defined medium, for 17 days.
Origin: Foreskin
The product was prepared and packaged using aseptic techniques. Store in an incubator at 37 °C, 5 % CO2 with saturated humidity.
Histology: HES stained paraffin section: Multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum.
6.5 cell layers
cell viability: 570 nm optical density, MTT test OD > 1.5 (CV = 1.3 %)
barrier function integrity test: exposure time inducing 50 % viability using Triton X-100 1 %: 4 <= ET50 <= 10 h: 6.6 h
biological safety: on blood of the donors was verified the absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs. On cells from the donors were verified the absence of bacteria, fungia and mycoplasma.
suggested expiration date: March 25, 2019
certified and released by Michel Bataillon, Quality Control Manager
Manufactured in accordance to the ISO9001 quality system of Episkin
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 16 μL
- Concentration: undiluted, used as supplied

NEGATIVE CONTROL
- Amount(s) applied: 16 µL

POSITIVE CONTROL
- Amount(s) applied: 16 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Test system

Details on study design:
Treatment
The test item was applied as supplied, at the dose of 16 µL to the epidermal surface of 3 living human skin models during 42 minutes at room temperature. To ensure a good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.

In the same experimental conditions, a positive control (16 µL of 5% sodium dodecyl sulfate - SDS) and a negative control (16 µL of distilled water – ADL Prochilab - Batch No. 180517) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBG6142V) in a 10 mL volumetric flask q.s. 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermises, during all the treatment period, the control items were recovered with a nylon mesh provided by Episkin SA.

Grading of reactions
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 (Dutscher - Batch No. 8041018). The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues. They were incubated for a 42 hours post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.

The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme is responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD are proportional to the number of living cells. The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours and 02 minutes at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours and 01 minute under gentle agitation in the dark, and the concentration of formazan was determined by measuring the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. The OD was measured in triplicate of MTT extract.

The measurement of OD at 570 nm of formazan extracts was performed in triplicate samples 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.

DATA EVALUATION
Viability (%): (mean OD test item / OD negative control) x100
The OD values obtained for each test item were used to calculate a percentage of viability relative to the negative control, which was arbitrarely set to 100%.

ACCEPTABILITY
- SD (standard deviation) <= 18%
- Negative control: OD value of the 3 replicates in the range >= 0.8 and <= 3.0, the optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range >=0.4 and <= 1.5 for the negative control.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
3.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.3 %
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none, the rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues
- Direct-MTT reduction:
The direct interaction of MTT with the test item was checked by adding 16 µl of the test item to 300 µl of the solution of MTT at 1 mg/ml (same condition as in the main test). A yellow solution was observed after 3 hours of incubation at approx. 37°C. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT:
The coloration potential of the test item in water was checked by adding 16 µl of the test item to 300 µl of distilled water. A colourless solution was obtained after 3 hours of incubation at approx. 37°C. The coloration potential of the test item in isopropanol was checked by adding 16 µl of the test item to 1.5 ml of isopropanol. A colourless solution was obtained after 2 hours of incubation at room temperature. Therefore the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean viability was 100 %, the mean OD for the negative control treated tissues was 0.909 and the standard deviation value of the viability was 12.6.
- Acceptance criteria met for positive control: yes, relative mean tissue viability for the positive control treated tissues was 1.3%, the mean OD was 0.012 and the standard deviation value of the viability was 0.1.
- Acceptance criteria met for variability between replicate measurements: yes, standard deviation calculated from individual tissue viabilities of test item treated tissues was 0.2.

Any other information on results incl. tables

Table 1: Individual and average values of OD after 42 minutes exposure

   Well ID  OD  Mean OD/ disc *  Mean OD/product  Viability (%)

Mean viability (%)**

 SD viability   Conclusion
Negative control  SPL1  0.992 / 1.031 / 1.022 1.015   0.909 111.6   100.0 12.6   -
  SPL 2  0.907 / 0.934 / 0.938 0.926  

 101.8

 

 

 

 

SPL 3 

0.783 / 0.785 / 0.793 

0.787

 

 86.5

 

 

 

Positive control 

SPL 4 

0.011 / 0.012 / 0.012 

 0.011

 0.012

 1.2

1.3

 0.1

 irritant

 

SPL 5

 0.014 / 0.012 / 0.012

 0.012

 

 1.3

 

 

 

 

SPL 6

0.013 / 0.014 / 0.014

 0.013

 

 1.4

 

 

 

Test item 

SPL 10

0.030/ 0.029 / 0.028

 0.029

 0.030

3.2

 3.3

 0.2.3 Irritant or corrosive
 

 SPL 11

 0.028 / 0.029/ 0.027

 0.028

 

3.1 

 

 

 

 

SPL 12

 0.033 / 0.032 / 0.032

 0.032

 

3.5

 

 

 

 * = mean of 3 values (triplicate of the same extract)

OD: optical density

SPL: sample

SD: standard deviation

Applicant's summary and conclusion

Interpretation of results:
other: additional in vitro corrosive test required to interprete the results
Conclusions:
The mean percent viablity of the treated tissues was 3.3 %, versus 1.3% of the positive control (5% Sodium Dodecyl Sulfate). The test item has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”.
Executive summary:

The aim was to evaluate the possible irritating effects of test item 2-hexyldecanoic acid after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model).

The test item 2 -hexyldecanoic acid was applied as supplied, at the dose of 16 µL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 42 hours and 00 minutes postincubation 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 experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 439 adopted 28 July 2015 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The mean percent viability of the treated tissues was 3.3, versus 1.3 % in the positive control (5% Sodium Dodecyl Sulfate).  

In accordance with the Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item 2 -hexyldecanoic acid has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”.