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Administrative data

Endpoint:
skin irritation / corrosion, other
Remarks:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 05 to 08, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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)
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Method B40bis, 2008
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Intercure 18
IUPAC Name:
Intercure 18
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Supplier: MatTek
- Tissue batch number(s): 23344
- Delivery date: 05 July 2016
- Date of initiation of testing: 05 July 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/ml
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

DECISION CRITERIA
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD of test item/mean OD of negative control ) x 100

The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control
The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg
Duration of treatment / exposure:
3 and 60 minutes
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes incubation
Value:
ca. 96.9
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes incubation
Value:
ca. 99.9
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was a white color. This was attributed to the intrinsic color of the test item and was considered not to have the potential to cause color interference.

Positive Control Item and Negative Control Item

The relative mean viabilities for each treatment group were as follows:

Percentage Viability:

Negative Control

3 minute exposure period = 100

60 minute exposure period = 100

Positive Control

3 minute exposure period = 3.0

60 minute exposure period = 3.0

Quality Criteria

The mean OD562 for the negative control treated tissues was 2.289 for the 3-Minute exposure period and 2.263 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 3.0 % relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
other: The substance is considered to be non-corrosive according to the OECD guideline 431
Conclusions:
Non-corrosive
Executive summary:

Method

The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes, according to the OECD guideline 431.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µl samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The percentage of tissue Viability , after incubation of the test substance, was:

3 minute exposure period = 96.9

60 minute exposure period = 99.6

Conclusion

The test item was considered to be non-corrosive to the skin.

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