Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Remarks:
Alfalfa, ext was observed to be a very thick dark coloured paste at room temperature.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 5.35 mg/L at 25C
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, tested neat
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Initially, the predictive capacity of the modified EpiDerm Skin Irritation Test (SIT) test method using MatTek EpiDermTM tissue model EPI-200 underwent full prospective validation from 2003-2007. The test method components of this method were used to define the essential test methods components of the original and updated ECVAM Performance Standards (PS).
A modification of the original EpiDerm SIT was validated using the original ECVAM PS in 2008. In 2008, ESAC concluded that the Modified EpiDerm SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number(s): 28672
- Production date: not available
- Shipping date: not available
- Delivery date: 04.12.2018
- Date of initiation of testing: 26.11.2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mg neat test material, 30 µL positive and negative control
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: not specified
- Incubation time: not specified
- Spectrophotometer:
- Wavelength: 570 nm
- Filter: none
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: not available

NUMBER OF REPLICATE TISSUES: 3 per condition

PREDICTION MODEL / DECISION CRITERIA
- A test item is considered an irritant (I) to skin in accordance with UN GHS Category 2 or EU R38 if the skin model viability after exposure and post-treatment incubation is =50%.
- A test item may be considered as a non-irritant (NI) if the skin model viability after exposure and post-treatment incubation is >50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg
Duration of treatment / exposure:
60 ± 1 minutes
Duration of post-treatment incubation (if applicable):
42 ± 4 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
95.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

See attached additional information on results.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results from the Reconstructed Human Epidermis test, conducted according to OECD test guideline 439 and to GLP, Alfalfa, ext.was classified as non-irritant to the skin.
Executive summary:

The Reconstructed Human Epidermis test, conducted according to OECD test guideline 439 and to GLP, was performed to evaluate skin irritation potential of Alfalfa, ext.

After 60-minute exposure on the surface of the EpiDerm constructed epidermis, and 42 ± 4 hours post exposure incubation time, viability of the tissue was assessed and compared to the negative (DPBS) and positive control (SDS). The percentage of viability obtained was 95.3% and therefore, Alfalfa, ext. was classified as non-irritant to the skin.