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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 - 24 Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
FREIE UND HANSESTADT HAMBURG, Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Test material

Constituent 1
Reference substance name:
Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol
EC Number:
286-056-9
EC Name:
Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol
Cas Number:
85186-72-7
IUPAC Name:
Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Justification for test system used:
This test method provides an in vitro procedure that, depending on information requirements, may allow determining the cytotoxic potency and skin irritancy of test items as a stand-alone replacement test within a testing strategy, in a weight of evidence approach. The test method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermal keratinocytes as cell source, representative tissue and cyto-architecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The procedure described under this test method allows the hazard identification of irritant substances in accordance with UN GHS category 1 or 2.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (EPI-200) MatTek In Vitro Life Science Laboratories, Bratislava II, Slovak Republic
- Tissue batch number(s): 23342
- Date of initiation of testing: 01 Jun 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C (for the first 35 min) and room temperature (for the second 25 min)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS) (not further specified)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Tecan Sunrise (Magellan Version 6.4)
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The optical density (OD) of the extraction solvent alone should be sufficiently small, i.e. OD <0.1. The tissue treated with the negative control (NC) should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.
- Barrier function: Concurrent negative (NC) and positive controls (PC) were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range
- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.
- Reproducibility: The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤18%.

NUMBER OF REPLICATE TISSUES: Triplicate tissues for test item, negative and positive control each

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Freeze-killed tissues
- Procedure used to prepare the killed tissues: The frozen tissues4 were stored in the freezer (-20 ± 5°C). The test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues.
- N. of replicates: Two treated and untreated freeze-killed tissues each
- Method of calculation used: True viability = (Viability of treated tissue – Interference from test chemical) = OD tvt – OD kt (where OD kt = (mean OD tkt – mean OD ukt)); tvt = treated viable tissue, kt = killed tissues, tkt = treated killed tissue, ukt = untreated killed tissue (NC treated tissue)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or is equal to 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritating to skin if the mean relative tissue viability of three individual tissues exposed to the test substance is above 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 µL
- Concentration: 100% (undiluted)

NEGATIVE CONTROL
- Amount(s) applied: 30 µL (D-PBS)
- Concentration: 100% (undiluted)

POSITIVE CONTROL
- Amount(s) applied: 30 µL SDS
- Concentration: 5%
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
triplicate tissues for each treatment and concurrent control groups

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
112.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
viability 100%
Positive controls validity:
valid
Remarks:
viability 9.6%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS
- Direct-MTT reduction: The test item was evaluated for the potential to interfere with the MTT assay reagent. Therefore, 30 μL of the test item were added to 1 mL of the MTT medium and incubated at 37°C, 5% CO2, and 95% humidity for 60 min. Untreated MTT solution was used as control. A discoloration of the test item to lilac was noted. Hence, due to interacting of the test item with the MTT measurement (reduction of MTT) an additional test with freeze-killed tissues had to be performed.
- Colour interference with MTT: Prior to the testing, the test item was evaluated for colour changes under aqueous conditions. Therefore, 30 μL of the test item was diluted in 300 μL deionized water and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 min. At the end of exposure time, the mixture was evaluated for the presence and intensity of the staining. No discolorations were noted.

ACCEPTANCE OF RESULTS
- Acceptance criteria met for negative control: The mean optical density (OD) of the negative control of 3 tissues was 1.417 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
- Acceptance criteria met for positive control: The viability of cells treated with the positive reference item, 5% SDS, was 9.6% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of all triplicates determined was below the limit of acceptance of 18%.

Any other information on results incl. tables

Table 1: Summarized results of in vitro skin irritation

 

Optical density (n = 3 tissues)

Standard deviation

Corrected OD (n = 3 tissues)

% Corrected OD / OD compared to the control

Negative control

1.417

0.015

-

100

Test item

1.684

0.107

1.597

112.7

Positive control

0.136

0.012

-

9.6

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
CLP: not classified

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