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Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-05 - 2017-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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:
adopted July 28, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted July 06, 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
N-butyltriphenylphosphonium bromide
EC Number:
918-205-7
Cas Number:
1779-51-7
Molecular formula:
C22H24P.Br
IUPAC Name:
N-butyltriphenylphosphonium bromide

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: three-dimensional reconstructed human epidermis model EpiDermTM, comprised of normal, human-derived epidermal keratinocytes
Cell source:
other: human
Justification for test system used:
recommended by guideline
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
EpiDermTM (EPI-200, Lot no. 25815) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Normal human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) were present under a functional stratum corneum. The Stratum corneum was multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of the cytotoxic marker substance sodium dodecyl sulphate (SDS). The barrier function is assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration. The containment properties of the model prevented the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure. The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The whole exposure period for the used EpiDermTM skin model was 60 minutes. The incubation conditions were 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.
- Temperature of post-treatment incubation (if applicable): 37°C

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)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The preferred assay for determining the magnitude of viability is the MTT assay. 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.
- Quality controls (QC) of the model: The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. The supplier of the test system determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results. A respective certificate of analysis regarding cell source, analysis for potential biological contaminants and analysis for tissue functionality and quality is added to the study report.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Not applicable. Prior to testing it was evaluated that there is no possible interference with the MTT measurement (OD 540 nm), due to colour changes or direct interacting with the MTT assay reagent.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA
mean tissue viability ≤ 50% Irritant (I); further testing is required to distinguish between Category 1 or Category 2
mean tissue viability > 50% non-irritant (NI)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 mg of test item were applied to the skin model and uniformly covered the skin surface with an area of 0.63 cm².

VEHICLE
- The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.

NEGATIVE CONTROL
- Amount(s) applied: 30 µL Dulbecco's phosphate buffered saline (D-PBS)
- Concentration (if solution): n.a.

POSITIVE CONTROL
- Amount(s) applied: 30 µL sodium dodecyl sulphate (SDS) solution
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
Posttreatment incubation period of the rinsed tissues in fresh assay medium of 42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value
Value:
64.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Viability: The preferred assay for determining the magnitude of viability is the MTT assay. 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute optical density (OD) of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5. The mean optical density (OD) of 3 negative control tissues was 1.794 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
- Acceptance criteria met for positive control: A 5% SDS (in H2O) solution was used as a positive control (PC) and tested concurrently with the test chemicals. Concurrent means here that the PC has to be tested in each assay, but only one PC is required per testing day. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%. The viability of cells treated with the positive reference item, 5% SDS, was 4.8% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
The standard deviation determined for all triplicates
- Acceptance criteria met for variability between replicate measurements: Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%. The standard deviation determined for all triplicates was below the limit of acceptance of 18%.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
n-Butyltriphenylphosphonium bromide was not irritating in the in vitro skin irritation reconstructed human epidermis model test.
According to the criteria of REGULATION (EC) No 1272/2008, n-Butyltriphenylphosphonium bromide does not have to be classified and has no obligatory labeling requirement for skin irritation.
According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations, n-Butyltriphenylphosphonium bromide does not have to be classified for skin irritation.