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:
9th October 2017 to 14th October 2017
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:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
MatTek EpiDerm™ MTT Viability Assay
Principles of method if other than guideline:
MatTek EpiDerm™ MTT Viability Assay
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
5-[4-Methoxy-2-(propan-2-yl)phenoxy]pyrimidine-2,4-diamine
Cas Number:
865304-71-8
Molecular formula:
C14H18N4O2
IUPAC Name:
5-[4-Methoxy-2-(propan-2-yl)phenoxy]pyrimidine-2,4-diamine
Test material form:
solid: crystalline
Details on test material:

Off-white

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm™ Tissue Samples
EpiDerm™ tissues, Lot 27154 Kit R, were received from MatTek on 10 Oct 2017 and refrigerated at 2-8ºC. Before use, the tissues were incubated (37oC ± 1oC, 5% ± 1% CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium before dosing.
Test Article Reduction of MTT
For each test article, 100 mg were mixed with 1 ml of MTT solution (1 mg/ml methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium [DMEM]). A negative control, undosed issues, was tested concurrently. The solutions were incubated at room temperature in the dark for 60 minutes. After incubation, the solutions were visually inspected for purple coloration, which indicates that the test article reduced MTT. Since tissue viability is based on MTT reduction, direct reduction by a test article can exaggerate viability, making a test article seem less irritating than its actual irritation potential. None of the test articles were found to have reduced MTT and the assay continued as per the protocol.
Dosing
At the request of the Sponsor, the test articles were dosed neat. For each test article, 100 mg were applied to the top of each EpiDerm™ tissue. Test article L-001739764-000M020 remained in contact with the EpiDerm™ tissue for 1, 4 and 24 hours. A negative control (undosed tissues) was tested in the same manner at 4 hours. A positive control (100 μl of 1% Triton® X-100) was tested at 4 and 9 hours. Each treatment with test article or control was conducted in duplicate.
Tissue Viability (MTT Reduction)
At the end of the selected exposure periods, each EpiDerm™ tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well plate containing 300 μl of MTT solution. The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then transferred to another 24-well plate containing 2.0 ml of extractant solution (isopropanol) per well. The tissues were then incubated overnight in the dark at room temperature. An aliquot of the extracted MTT formazan was measured at 540 nm using a plate reader (μQuant Plate Reader, Bio-Tek Instruments, Winooski, VT), subtracting the absorbance at a reference wavelength of 690 nm.
Control samples:
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
100 mg
Duration of treatment / exposure:
The solutions were incubated at room temperature in the dark for 60 minutes.
Duration of post-treatment incubation (if applicable):
At the end of the selected exposure periods, each EpiDerm™ tissue was rinsed with phosphate
buffered saline (PBS) and transferred to a 24-well plate containing 300 μl of MTT solution. The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then transferred to another 24-well plate containing 2.0 ml of extractant solution (isopropanol) per well. The tissues were then incubated overnight in the dark at room temperature.
Number of replicates:
Each treatment with test article or control was conducted in duplicate.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
> 24
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The ET50 score of the positive control, 1% Triton® X-100, was 6.0 hours, which met MatTek's acceptance criterion of 4.8-8.7 hours.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The ET50 (hr) of L-001739764-000M020 exposed for 1, 4 and 24 hours was >24.0, which gave an irritancy classification of Non-Irritating
Executive summary:

MatTek EpiDerm™ tissue samples were treated in duplicate with the test articles, and positive control for various exposure times listed below. Negative controls (undosed tissues) were tested at 4 hours only. Following treatment, the viability of the tissues was determined using methyl thiazole tetrazolium (MTT) uptake and reduction. The absorbance of each sample was measured at 540 nm using a reference wavelength of 690 nm. The viability was then expressed as a percent of negative control values. The mean percent viability for each time point was used to calculate an ET50, which represents the time at which the EpiDerm™ tissue viability was reduced 50% compared to control tissues. The ET50 scores were converted to an irritancy classification. 


The ET50 (hr) of L-001739764-000M020 exposed for 1, 4 and 24 hours was >24.0, which gave an irritancy classification of Non-Irritating