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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:
05 - 11 October 2018
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:
2018
Report Date:
2018

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: 05347/MA
- Expiration date of the lot/batch: Retest date June 2022
- Storage condition of test material: room temperature, darkness
- Purity: >99% (GC)
- Purity test date: 31 August 2018

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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin SA, RHE/S/17
- Tissue batch number: 18-RHE-118
- Production date: cells are grown on inert polycarbonate filters in chemicaly defined medium, for 17 days
- Delivery date: 09 October 2018
- Date of initiation of testing: 09 October 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washed with 25 x 1 ml of DBPS (Dutscher - Batch No. 4090718)
- Observable damage in the tissue due to washing: rinsed tissues were checked of any coloration and noted to be whitish, comparable coloration to that of the negative control tissues
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800 absorbance microplate reader supplied by BioTek (controlled every year and calibrated if necessary) with validated software Gen5 ELISA V1.05.11 supplied by BioTek
- Wavelength: 570 nm
- Filter: no use of filter mentioned
- Linear OD range of spectrophotometer: not mentioned

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.2 (CV = 3.6%), MTT test at 570 nm optical density, specification criteria: OD: > 0.7)
- Barrier function: Exposure time inducing 50% viability using Triton X-100 1% = 4.0 h (Specification criteria: 4.0 <= ET50 <= 10.0 h)
- Morphology: histological observation of HES stained vertical paraffin sections: 7 cell layers (specification criteria: number of cell layers >= 4), absence of significant histological abnormalities, well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: on blood of donor the following was verified: absence of HIV1 and 2 antibodies, absence of hepatits C antibodies, absence of hepatitis B antigen HBsp;
on epidermal cells of the donor the following was verified: absence of mycoplasma
- Reproducibility:within range of historical controls

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- Classification of irritation potential is based upon relative mean tissue viability following the 42-minute exposure period followed by the 42-hour post-exposure incubation period according to:
Relative mean tissue viability is <= 50%: UN GHS Category 2 "irritant" or UN GHS Category 1 "corrosive" in absence of information on a skin corrosion test
Relative mean tissue viability is > 50%: non-irritant UN GHS no category)
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:
MAIN TEST
Application of test item and rinsing
Exposure: 42 min at room temperature, to ensure a good contact with the epidermises, during all the treatment period, the test and control items were recovered with a nylon mesh provided by Episkin SA
Amount applied: 16 µl
Negative control: 16 µl distilled water
Positive control: 16 µl 5% w/v SDS
Washing: 25 x 1 ml DPBS
Post-exposure incubation: 41 h and 55 min at 37°C

MTT Loading/Formazan Extraction
MTT solution: 300 µl of 1.0 mg/ml per well
Incubation: 3 h at 36.5 and 38 °C
The precipated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was determined by measuring the OD at 570 nm in triplicate, just after dilution of the extracts 1:2 in isopropanol.

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:
94
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.3 %
Remarks on result:
no 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 sollution 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, mean OD for the negative control treated tissues was 0.894 and the standard deviation value of the viability was 3.5%
- Acceptance criteria met for positive control: yes, relative mean tissue viability for the positive control treated tissues was 1.3% relative to the negative control treated tissues 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 9.3%

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  0.908 / 0.894 / 0.891 0.897   0.894 100.4   100.0 3.5   -
  SPL 2  0.937 / 0.926 / 0.908 0.923    103.3      
  SPL 3  0.867 / 0.840 / 0.878  0.861    96.3      
Positive control  SPL 4  0.011 / 0.014 / 0.013   0.012  0.012  1.3 1.3  0.1  irritant
  SPL 5  0.012 / 0.011 / 0.011  0.011    1.2      
  SPL 6 0.013 / 0.013 / 0.014  0.013    1.5      
Test item  SPL 7 0.917 / 0.939 / 0.941  0.932  0.840 104.3  94  9.3  non irritant
   SPL 8  0.772 / 0.764 / 0.775  0.770   86.2       
  SPL 9  0.816 / 0.832 / 0.807  0.818   91.5      

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

** = the mean viability of the negative control tissues is set as 100%

OD: optical density

SPL: sample

SD: standard deviation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The mean percent viablity of the treated tissues was 94.0 %, versus 1.3% of the positive control (5% Sodium Dodecyl Sulfate). The test item has to be considered as non-irritant to skin.
Executive summary:

The aim was to evaluate the possible irritating effects of test item Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model).

The test item Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic 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 41 hours and 55 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 94.0%, versus 1.3 % in the positive control (5% Sodium Dodecyl Sulfate).  

In accordance with the Regulation EC No. 1272/2008, the test item Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.  No hazard statement or signal word is required.