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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-12-10 - 2019-02-20 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
OECD Guidelines for the Testing of Chemicals Part 431, adopted 29. Jul. 2016
“In vitro Skin Corrosion: reconstructed human epidermis (RHE) test method”
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Commission Regulation (EU) No.440/2008, EU-Method B.40 BIS. “IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST“ dated 30. May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany

Test material

Constituent 1
Reference substance name:
Yucca schidigera, ext.
EC Number:
607-033-5
Cas Number:
223749-05-1
IUPAC Name:
Yucca schidigera, ext.
Test material form:
solid: particulate/powder
Remarks:
fine beige
Details on test material:
Storage: Room Temperature (20 ± 5 °C)
Homogeneity: homogeneous
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel at room temperature (20±5°C).

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: not applicable
Justification for test system used:
First step in top-down approach; in vitro test as indicated in the REACH Regulation.
Vehicle:
water
Remarks:
Defined amounts of the test item were applied together with 25 µL demineralised water.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The test system is a commercially available EpiDermTM-Kit, procured by MatTek. The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.
- Tissue batch number(s): 28675, 28683
- Delivery date: 11. Dec. 2018 (EPI-200-SIT, Batch 28675), 12. Feb. 2019 (EPI-200-SCT, Batch 28683)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The inserts were thoroughly rinsed with DPBS.
- Observable damage in the tissue due to washing: none stated
- Modifications to validated SOP: none stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of 2 mL in the freezer (– 20±5°C).
One aliquot of the stock solution was thawed and diluted with 8 mL of medium. This MTT-solution with the resulting concentration of 1 mg/mL was used in the test.
- Incubation time: 3h
- Spectrophotometer: Anthos Reader 2010 Flexi
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 per test

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In addition to the normal test procedure described, the functional check employed two freeze-killed tissues treated with the MTT reducing test item (for 3 minutes and 1 hour experiment) and two untreated killed tissues (for 3 minutes and 1 hour experiment) that showed the small amount of MTT reduction due to residual NADH and associated enzymes within the killed tissues
- Procedure used to prepare the killed tissues (if applicable): Freeze killed tissues were prepared by placing untreated tissues in freezer (– 20 ± 5°C) over night. Once killed, the tissues were stored indefinitely in the freezer.
- N. of replicates : 2
- Method of calculation used: The mean value “% Viability (freeze-killed-tissues)” of the additional test was subtracted from the mean value “% Viability” of the main test to achieve the corrected tissue viability.

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 viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): ca. 25 mg test item were applied together with 25 µL demineralised water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL demineralised water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL potassium hydroxide solution (8 M)
Duration of treatment / exposure:
3 min or 1 h
Duration of post-treatment incubation (if applicable):
Incubation with MTT solution for 3 hours
Number of replicates:
2 x 2 tissues

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item, mean, 3 min incubation
Value:
91.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item, mean, 1 h incubation
Value:
69.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none stated
- Direct-MTT reduction: The resulting solution was colourless, therefore no binding capacity had to be tested.
- Colour interference with MTT: The MTT solution changed its colour to purple within 1 hour, so the test item had shown its ability to reduce MTT

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The validity of the skin corrosion study at LAUS GmbH was demonstrated in a proficiency study. For this purpose, 12 proficiency chemicals (indicated by the OECD 431 guideline) were tested.
All of the 12 proficiency chemicals were correctly categorized.
Therefore, the proficiency of the skin corrosion study was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
non-corrosive
Conclusions:
The study was conducted under GLP according to OECD guideline 431 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. The validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the corrosive potential of the test substance to the skin in vitro.
The mean value of relative tissue viability of the test item was reduced to 85.9% after 3 minutes treatment. This value is above the threshold for corrosivity (50%). After 1 hour treatment, the mean value of relative tissue viability of the test item was reduced to 56.4%, lying above the threshold for corrosivity (15%). Therefore, the test item is considered as non-corrosive to skin.
In a tiered in vitro testing strategy, this result (non-corrosive) may however only give an indication of the skin-damaging potential, but does not allow a definitive classification according to Regulation 1272/2008. So, an additional skin irritation test should be conducted.
Executive summary:

One valid experiment of the main test was performed according to OECD TG 431 under GLP.

Two experiments of the additional test were performed.

The first experiment of the additional test was not valid, because the standard deviation of the replicates of the test item in the 1 hour experiment was 61.2%, required is ≤30%. The first experiment of the additional test is not reported in this report, but the raw data are kept in the test facility in the GLP- archive.

The second experiment of the additional test was valid and the results are reported here.

 

In the pre-test the test item showed MTT-reducing properties. The probability to influence the photometric measurement had to be excluded. Therefore, an additional test using freeze-killed tissues that possess no metabolic activity, but absorb and bind the test item like viable tissues was performed. The result of the additional test showed that MTT reduction by the test item did not influence the result of the study.

In the main test two tissues of the human skin model EpiDerm were treated with the test item for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

 

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.7 (3 minutes experiment) and 1.6 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 5.7% for the 1 hour treatment.

After 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 85.9%. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, mean value of relative tissue viability was reduced to 56.4%. This value, too, is above the threshold for corrosion potential (15%).

 

Therefore, the test item Yucca schidigera, ext. is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.