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:
From July 20 to 27, 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Acid Red 310 RC
IUPAC Name:
Acid Red 310 RC
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: normal human-derived epidermal keratinocytes
Vehicle:
other: PBS (phosphate-bufferd saline)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, USA)
- Tissue batch number(s): 23347- Tissue surface: 0.63 cm²The EpiDerm™ tissues are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media

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

REMOVAL OF TEST MATERIAL AND CONTROLS
The tissues were rinsed with PBS, blotted to remove the test substances, and then transferred to fresh medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3h
- Spectrophotometer: Libra S22
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:

3CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Tissues: freeze-killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates : 2
- Method of calculation used: True viability = %Viability of treated tissue – (%)NS

MTT NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:PREDICTION MODEL / DECISION CRITERIA
The cut-off values for the prediction of irritation are given below; these values are stated in OECD Test Guideline No. 439 (1), par. 36:
-In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS (3) Category 2.
-The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%. A single testing run composed of three replicate tissues should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5%, a second run should be considered, as well as a third one in case of discordant results between the first two runs.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg
Duration of treatment / exposure:
one hour
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 75.7
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Direct MTT reduction-functional check in tubes
25 mg of the test substance was added to 1.0 mL of MTT medium. Solution was incubated for 1 hour (37 ± 1°C, 5 ± 1 % CO2, humidified). The test substance changed colour of MTT medium to red. The test substance is not directly-reducing.

Colour interference
25 mg of the test substance was added to 2 mL of isopropyl alcohol. The test substance was insoluble in it. After 125 minutes of shaking at room temperature, mixture was centrifuged; supernatant was decanted and used for measuring of spectra in range of wavelength from 190 to 900 nm.The test substance almost did not absorbed light in range 540-600 nm; OD570 were 0.0-0.008. Nevertheless, colorant control was also included in the test because enough tissues were available.

MTT test
25 mg of the test substance was placed directly atop the tissue moistened with 25 µL of PBS. The material was then spread on the tissue surface. Length of exposition was 60 minutes of which 25 minutes were tissues placed at room temperature and the remaining 35 minutes at 37±1°C, 5±% CO2. After that the test substance was removed, tissues were rinsed and post-incubated. 1st post-incubation took 24 hours, 2 minutes and 2nd took 18 hours, 39 minutes. Many washing cycles must have been performed due to removal of the test substance from tissue surface. Nevertheless, tissues (and walls of inserts) remained coloured even after extraction with isopropyl alcohol, what means, that the test substance is insoluble in it. After the first post incubation medium was yet slightly coloured with the test substance. After post-incubations the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg•mL-1). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol for 2 hours, room temperature and shaking, and the optical density of the extracted formazan was determined using a spectrophotometer at λ=570 nm. In addition, two viable tissue replicates, which underwent the entire testing procedure (treatment for 60 minutes) but were incubated with assay medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSCliving) control, were used concurrently with MTT test. OD570 of these tissues were 0.017 and 0.008 so average value was 0.013, what is 0.7 % of negative control OD570.

Data correction for colour interference:
True viability = % Viability of treated tissue – % NSCliving 75.7 % - 0.7 % = 75.0 %

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Not irritant
Executive summary:

Method

The test item was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No.439.

After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. Length of exposition was 60 minutes. Three tissues were used for the test substance and every control. Two tissues more were used as colorant control to correction of possible colour interference, which undergo the entire testing procedure excepting of incubation with MTT medium.

After removal of the test substance, tissues were post-incubated for approximately 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

 

Results

Under the above-described experimental design average viability of treated tissues was 75.7 % (75.0 % after correction).

 

Conclusion

Not irritant.

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