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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 2017-05-24 to 2017-06-09
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trihydrogen [29H,31H-phthalocyaninetrisulphonato(5-)-N29,N30,N31,N32]cuprate(3-), compound with N,N'-di(o-tolyl)guanidine (1:3)
EC Number:
277-086-3
EC Name:
Trihydrogen [29H,31H-phthalocyaninetrisulphonato(5-)-N29,N30,N31,N32]cuprate(3-), compound with N,N'-di(o-tolyl)guanidine (1:3)
Cas Number:
72928-60-0
Molecular formula:
C32H16-τN8Cu(SO3C15H18N3)τ
IUPAC Name:
Bis and tris and tetra [N-(2-methylanilino)-N’-(2-methylanilino) methaniminium][phthalocyaninesulfonato-κ4N29,N30,N31,N32]cuprate(II)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell source:
other: Episkin SA, RHE/S/17 (batches 17-RHE-064, Episkin SA, RHE/S/17 and 17-RHE-059)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin SA, RHE/S/17

- Tissue batch number(s): 17-RHE-064 and 17-RHE-059
- Delivery date: 2017-06-07


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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength: 570 nm





CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: 2 living and 2 killed Human skin model surfaces
- Procedure used to prepare the killed tissues : The 2 additional killed Human skin model surfaces (Episkin SA, RHE/S/17 Batch
No. 17-RHE-059) were defrozen the day of the treatment. 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 1 mL of growth medium during 2 hours and 05 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
- N. of replicates : 2
- Method of calculation used: As the test item is identified as producing both direct MTT reduction and colour interference:
True viability % =
[(OD of living tissues exposed to test item - OD of killed tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT + OD of killed tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 100



PREDICTION MODEL / DECISION CRITERIA
Following the guideline TG 439
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg


NEGATIVE CONTROL
DPBS

POSITIVE CONTROL
- Concentration (if solution): 5% SDS prepared by weighing 0.5 g of SDS on a 10 mL flask and filled up to 10 mL with distillated water
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Reconstructed human epidermis: 3 replicates for test item, positive and negative controls

Living human skin model surfaces (Non specific colour controls or NSC): 2 replicates
killed human skin model surfaces (Non specific colour controls or NSC): 2 replicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 84.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:

The test item is considered to be non irritant to skin:
- if the viability after 42 minutes of exposure and 42 hours of post-treatment incubation is > 50%.

The test item is considered to be irritant to skin:
- if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%.
In accordance with the Regulation EC No. 1272/2008, the item is to be classified in Category 2 “Irritant”.
The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

Any other information on results incl. tables

  number of replicates Mean OD / product mean viability (%) SD Conclusion
Negative control 3 0.895 100.0 6.7  
Positive control 3 0.013 1.5 0.3 Irritant
Test item 3 0.814 91.0 5,1  
Test item NSC Control living tissues 2 0,06 6,6 0,4  
Test item NSC Control killed tissues 2 0,036 4 3,8  
Test item killed tissues MTT control 2 0,35 3,9 1,6  
Test item corrected     84,5   non irritant 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In accordance with the Regulation EC No. 1272/2008, the test item has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.
Executive summary:

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

The test item was applied at the dose of 16 mg, 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 05 minutes post-incubation 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. Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific living and killed colour controls. Moreover, 2 living and 2 killed Human skin model surfaces were treated in the same manner but they were incubated in culture growth medium instead of MTT solution in order to generate non-specific living and killed colour controls. 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 corrected percent viability of the treated tissues was 84.5%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation EC No. 1272/2008, the test item has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

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