Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8th april 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The In vitro Skin Corrosion: Human Skin Model Test is based on the observation that skin corrosion (necrotic damage of viable skin cells) shows a high correlation with skin cell cytotoxicity, occurring rapidly after brief exposure of the skin barrier (stratum corneum) to a corrosive chemical. It is designed to predict and classify the skin corrosivity potential of a chemical by using a three-dimensional human epidermis model. The epidermis model (e.g. EpiDermTM) is derived from human keratinocytes and consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK, which are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation at the cutting edge of in vitro skin technology. Ultrastructurally, the skin models closely parallel human skin. The In vitro Skin Corrosion: Human Skin Model Test consists of topical application of the test material to the tissue for 3 minutes and 1 hour, followed by immediate determination of the cytotoxic effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the exposure period.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
White powder
Specific details on test material used for the study:
batch 18114773 - expiry date : oct 2018

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Vehicle:
water
Details on test system:
Test Item Preparation :
25 μL of deionised water and 25 ± 2mg (39.7 mg/cm2) of the test item were applied onto the surface of duplicate EpiDermTM tissue.

Test System :
1- Epi-200 Kit Components Needed for the Assay
EpiDerm™ Kit Lot No.: 25806
1 Sealed 24-well plate Contains 24 inserts with EpiDerm™ tissues on agarose
2 24-well plates For MTT viability assay
4 6-well plates For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium DMEM-based medium
1 bottle DPBS Rinse Solution For rinsing the inserts in MTT assay

2- MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant Solution (Isopropanol) For extraction of formazan crystals
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
Amount/concentration applied:
negative control : 50µl
positive control : 50µl
test item 25mg
Duration of treatment / exposure:
3min and 60 min
Duration of post-treatment incubation (if applicable):
60

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: % relative absorbance (% negative control)
Run / experiment:
3 min
Value:
ca. 103.1
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: % relative absorbance (% negative control)
Run / experiment:
60 min
Value:
ca. 90.6
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
This in vitro study was performed to assess the corrosive potential of 1574 ACIDE CRIST by means of the Human Skin Model Test with EpiDerm™ tissues models.
The test item passed the MTT- and the colour interference pre-tests.
Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.
Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 19.5 hours at room temperature.
The required acceptability criteria were met.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the reported experimental conditions, 1574 ACIDE CRIST is non corrosive to skin according to EU CLP and UN GHS.
Executive summary:

This in vitro study was performed to assess the corrosive potential of 1574 ACIDE CRIST by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the relative absorbance value did not decrease (103.1%) after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 90.6%. Both values did not affect the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item 1574 ACIDE CRIST is non corrosive to skin according to EU CLP and UN GHS