Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2020 to 15 October 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
ethyl (2E)-3-(cyclopropylamino)-2-[(2,4-dichloro-3-methylphenyl)carbonyl]prop-2-enoate
Molecular formula:
C16H17Cl2N1O3
IUPAC Name:
ethyl (2E)-3-(cyclopropylamino)-2-[(2,4-dichloro-3-methylphenyl)carbonyl]prop-2-enoate
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Remarks:
Episkin SA, RHE/S/17 Batch No. 20-RHE-133
Source species:
human
Cell type:
other: reconstructed epidermis of normal human keratinocytes
Cell source:
other: Foreskin, number of donors not specified
Source strain:
other: BATCH 20-RHE-133
Justification for test system used:
EpiSkin (VRM) is specified in the OECD Guidelines 439 and has undergone a validation study from 2003 to 2007 and was used to develop the original PS. Thus it was considered a adequate in vitro test system
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE/ Human Epidermis RHE/S/17
- Tissue batch number(s): 20-RHE-133
- Production date: 13 October 2020
- Shipping date: 13 October 2020
- Delivery date: 13 October 2020
- Date of initiation of testing: 13 October 2020

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

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

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: 3hours and 4 minutes
- Spectrophotometer: ELx800 absorbance microplate reader BioTek, Software Gen5 ELISA V1.05.11, BioTek.
- Wavelength: 570nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: mean viability OD 1.3 (specification O.D. > 0.7)
- Barrier function: 7.4 h (specification 4.0h≤ET50 ≤ 10.0h)
- Morphology: 6.5, basal, spinous, granular layers and a multilayerd stratum corneum
- Contamination: no
- Reproducibility: Yes

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non-corrosive” OR if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.
- The test substance is considered to be non-corrosive to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
other: NSC living control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% sodium dodecyl sulfate

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
41 hours and 50 minutes
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
91.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean viabilityh 100%
Positive controls validity:
valid
Remarks:
Mean viability: 1.9% Conclusion: Irritant
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: In 2019 the lab conducted more than 60 studies with different test items according to OECD 439 guideline and the same test system, prooving their technical proficiency

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, 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. Mean OD of negative control is 0.948
- Acceptance criteria met for positive control: Yes Mean Viability < 40%, and SD value of the % viability ≤ 18%. Viability of positive control is 1.9%
- Acceptance criteria met for variability between replicate measurements: Yes, SD ≤ 18% SD of negative 8,8, positive control 0.3 and test item 14.3
- Range of historical values if different from the ones specified in the test guideline: The values were in range of historical data of the facility

Any other information on results incl. tables








































































































 



Well ID



OD



Mean OD /   disc (#)



Mean OD / product



Viability


%



Mean


viability


%



SD



Conclusion



Negative control



SPL 1



0.862 0.863


0.867



0.864



0.948



91.2


108.7


100.1



100.0



8.8



 



SPL 2



1.052 1.030


1.010



1.030



SPL 3



1.041 0.944


0.862



0.949



Positive control



SPL 4



0.017 0.017


0.020



0.018



0.018



1.9


1.7


2.2



1.9



0.3



Irritant



SPL 5



0.016 0.017


0.016



0.016



SPL 6



0.022 0.021


0.021



0.021



Test item


PH-20/0589



SPL 10



1.136 1.010


0.920



1.022



0.871



107.8


80.3


87.5



91.9



14.3



 



SPL 11



0.834 0.718


0.733



0.761



SPL 12



0.902 0.724


0.862



0.829



Test item


 PH-20/0589


NSC living control



SPL 18



0.003 0.003


0.004



0.003



0.003



0.3


0.3



0.3



0.0



SPL 19



0.004 0.004


0.003



0.003



Test item


 PH-20/0589 corrected



 



 



 



 



91.6



 



Non irritant


Applicant's summary and conclusion

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Remarks:
not irritating according to EU criteria
Conclusions:
The test item is not irritant to skin
Executive summary:

The skin irritating properties of the test item were determined according to OECD 439 under GLP conditions. The test item was directly applied to EpiSkin SkinEthic RHE/Human Epidermis RHE/S/17 consisting of 6.5 cell layers (basal, spinous and granular layers and a multilayered stratum corneum). After initial exposure of three replicates with the test item of 42 minutes the test system was washed 25 times with 1ml DPBS and subseqently incubated for 41 hours and 50 minutes @ 37C and 5% CO2. Following contact with MTT solution the cell viability was assessed by extracting the formazan crystal from the tissue and its optical density measured. Preliminary testing showed that the test item causes colour interference and thus, next to a positive control (SDS 5%) and a negative control (Aq.dest) a NSC living control with the test item underwent the entire test procedure but not incubated with MTT to generate a non-specific colour control. The positive and negative control all fullfilled the acceptability criteria. The SD of all samples fell into the range of ≤ 18% and thus fullfilled the acceptability criteria. The test item has to be considered as Non-irritant to skin corresponding to UN GHS No Category.

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