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:
04.03.2021-25.03.20221
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:
Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction
EC Number:
953-265-8
Molecular formula:
Not applicable
IUPAC Name:
Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Remarks:
(SkinEthic RHE® model)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Justification for test system used:
The SkinEthic RHE® model has been validated for irritation testing and its use is recommended by
the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be
suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 21-RHE-035
- Production date: N/A
- Shipping date: 23.03.2021
- Delivery date: 23.03.2021
- Date of initiation of testing: 23.03.2021

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

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: no
- Modifications to validated SOP: no

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 and 5 minutes
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.4 (Acceptance criterion: >0.7). Historical negative control mean OD range = 0.787-1.130 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.5)
- Barrier function: 6.5 h (Acceptance criterion: 4.0h ≤ ET50 ≤10.0h)
- Morphology: 5.5 Cell layers (specification ≥ 4). Multi-layered, highly differenciated epidermis consisting of organized basal, spinous and granular layers and a multilayered stratum corneum.
- Contamination: No

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The direct interaction of MTT with the test item was checked by adding 16 mg of the test item to 300 μL of the solution of MTT at 1 mg/mL. A purple solution was observed after 3 hours of incubation between 37.1°C and 37.5°C, 5% CO2.
Therefore, the test item was identified as a direct MTT reducer: two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non-specific MTT reduction control.

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

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered as non-irritant to skin if the mean percent viability after 42 minutes
exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2) 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”.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) 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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32 mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
42 min at room temperature.
Duration of post-treatment incubation (if applicable):
41-hour and 36 minutes
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
91.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
distilled water
Positive controls validity:
valid
Remarks:
5% SDS
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was
performed for the EpiSkin model, plus a reduced validation with the SkinEthic RHE model, having
into account that both models are very similar. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean OD = 0.950 (OD measured after 1:2 dilution
in isopropanol; criterion for acceptability should be in the range ≥ 0.4 and ≤1.5).
- Acceptance criteria met for positive control: yes, mean viability = 1.3% (criterion for acceptability
should be < 40%).
- Acceptance criteria met for variability between replicate measurements: yes. SD of negative, positive and test item replicates were 10.4, 0.1 and 15.3% respectively (criterion for acceptability, SD ≤ 18%).

Any other information on results incl. tables

Table 1. Table of results













































































































 Well IDODMean OD/disc (#)Mean OD/productViability %Mean viability %SD viabilityConclusion
Negative controlSPL-1

1.061


1.073


1.046


1.0600.950111.6100.010.4 
SPL-2

0.779


0.886


0.945


0.87091.6
SPL-3

0.948


0.891


0.920
0.92096.8
Positive controlSPL-4

0.012


0.012


0.013
0.0130.0121.41.30.1Irritant
SPL-5

0.011


0.010


0.011
0.0111.2
SPL-6

0.012


0.012


0.012
0.0121.3
Test item PH-21/0194SPL-7

1.070


1.074


1.098
1.0810.913113.896.115.3 
SPL-8

0.848


0.828


0.845
0.84188.5
SPL-9

0.821


0.811


0.820
0.81886.1

PH-21/0194


NSMTT
SPL-10

0.042


0.042


0.042
0.0420.0414.44.30.1 
SPL-11

0.040


0.040


0.040
0.0404.2
PH-21/0194 corrected 91.8 Non-irritant

#: mean of 3 values (triplicate of the same extract)
SPL: sample
OD: optical density
SD: standard deviation

Applicant's summary and conclusion

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The test item can be considered as not irritant to skin as the mean percent viability of the treated tissues was found 91.8% in the in vitro RHE test.
Executive summary:

An in vitro skin irritation test was conducted for the test item in a reconstructed human epidermis model ( SkinEthic™) according to OECD TG 439 (GLP study). Three epidermis units, previously moistened with 10 μL of distilled water, were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated for the post-treatment incubation period for 41-hour and 36 minutes in fresh medium at 37ºC, 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Under the test conditions, the mean percent viability of the treated tissues was 91.8%, versus 1.3% in the positive control (5% SDS). Therefore, the test item is considered as not irritant to the skin.