2-Chloro-8-((S)-1-cyclopropyl-ethyl)-6-[(5-methanesulfonyl-pyridin-2-ylmethyl)-amino]-8Hpteridin-7-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 - 21 November 2016

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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Remarks:

44 hours extraction period, not overnight as specified on protocol. Also plates were not shaken for 15 minutes at the end of the extraction period. These deviations did not affect the integrity or outcome of the study as all acceptance criteria were met.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-chloro-8-[(1S)-1-cyclopropylethyl]-6-{[(5-methanesulfonylpyridin-2-yl)methyl]amino}-7,8-dihydropteridin-7-one

EC Number:

825-281-4

Cas Number:

1817775-66-8

Molecular formula:

C18H19ClN6O3S

IUPAC Name:

2-chloro-8-[(1S)-1-cyclopropylethyl]-6-{[(5-methanesulfonylpyridin-2-yl)methyl]amino}-7,8-dihydropteridin-7-one

In vitro test system

Test system:

human skin model

Remarks:

EpiDermTM SIT (EPI-200)

Source species:

human

Cell type:

other: comprising a reconstructed epidermis with a functional stratum corneum

Vehicle:

unchanged (no vehicle)

Details on test system:

ASSESSMENT OF MTT INTERACTING SUBSTANCES
In order to assess the potential non-specific reduction of the test article, a dose of 25 mg of
test article was added to 1 mL of MTT and colour change assessed after 60 minutes. There
was no change in colour therefore the test article did not interact with MTT.

ASSESSMENT OF STAINING POTENTIAL FOR NON-COLOURED
SUBSTANCES
In order to assess the potential of staining, 25 mg of the test article was added to 0.3 mL
deionised water and 0.3 mL isopropanol in a glass container and incubated for 60 minutes at
37±1ºC, 5±1% CO2. At the end of the incubation, the mixture was shaken and any colour
change assessed. There was no change in colour therefore the test article did not have the
potential to stain the tissue.

APPLICATION OF TEST AND CONTROL SUBSTANCES
EpiDermTM SIT (EPI-200) tissues were kept in their packaging until the next step. The tissues
were set up the day prior to treatment by placing each tissue onto 0.9 mL maintenance
medium (supplied with the EpiDermTM SIT (EPI-200) tissues) in 6-well plates and incubating
at 37°C.
The test was performed on a total of three tissues per test article, negative and positive
control.
25 mg of the test article was added topically to the tissues. The tissues were moistened with
25 μL of PBS, prior to application. A volume of 30 μL of the positive and negative control
solutions was used.
The treated tissues were paced into an incubator at 37±1ºC, 5±1% CO2 for 35 minutes. The
plates were removed from the incubator and placed into a sterile hood until the 60 minute
treatment period was complete for each tissue. Following treatment, substances were
removed by washing the tissues. The tissues were then placed on the appropriate medium and
incubated for 42 hours 35 minutes.

CELL VIABILITY MEASUREMENTS
Upon completion of the 42 hour recovery period, each tissue was rinsed with PBS before
being placed on 0.3 mL of 1 mg/mL MTT in PBS and incubated for three hours at 37°C.
After incubation, any resultant colour was extracted. Each tissue was flooded with 2 mL
isopropanol, the plate was sealed and then left at room temperature for approximately
44 hours (protected from light). See Section 6 for details of Protocol Deviations.
Upon completion of the extraction each tissue was pierced using a hypodermic needle and the
extract drained and placed into a 96 well microtitre plate. The optical density of each
resultant extract was determined spectrophotometrically at 570 nm, using extraction solvent
as a blank and cell viability was calculated for each tissue as a percentage of the mean of the
negative control tissue.

Assay Acceptance Criteria
The assay was considered valid if the following criteria were met:
1. The absolute OD570 of the negative control tissues in the MTT test is an indication of the
tissue viability in the testing laboratory after the shipping and storage procedure and
under specific conditions of the assay. The OD values for the negative controls should be
between ≥0.8 and ≤2.8 for this tissue model
2. The viability of the tissues treated with the positive control should be ≤20%
3. The standard deviation (SD) for tissues should be ≤18%.

Interpretation of Results
Test substances (test articles) are considered to be irritant to skin in accordance with UN
GHS Category 2 if the tissue viability after exposure and post-treatment incubation is less
than or equal to 50%, relative to the negative control.
Test substances (test articles) are considered to be non-irritant to skin in accordance with UN
GHS No Category if the tissue viability after exposure and post-treatment incubation is
greater than 50%, relative to the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

25 mg of test article
30 μL of the positive and negative control

Duration of treatment / exposure:

The treated tissues were paced into an incubator at 37±1ºC, 5±1% CO2 for 35 minutes. The
plates were removed from the incubator and placed into a sterile hood until the 60 minute
treatment period was complete for each tissue.

Duration of post-treatment incubation (if applicable):

Following treatment, substances were removed by washing the tissues. The tissues were then placed on the appropriate medium and incubated for 42 hours 35 minutes.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 99.6

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

3.4%

Applicant's summary and conclusion

Interpretation of results:

other: not classified acc. CLP

Conclusions:

This study was conducted to determine whether the test article causes irritation in the in vitro
skin model EpiDermTM SIT (EPI-200).
EpiDermTM SIT (EPI-200) inserts were treated with BI 740293, negative control (phosphate
buffered saline (PBS)) and positive control (5% w/v sodium dodecyl sulphate (SDS)) for
60 minutes. At the end of the treatment period, the tissues were washed with PBS and cell
viability was assessed using the MTT assay. The skin irritation potential was classified
according to the remaining cell viability obtained after test article treatment.
The group mean viability for the test article was 99.6%, for the negative control was 100.0%
and for the positive control was 3.4%.
The test article, BI 740293, was not considered to be irritant in the in vitro skin model
EpiDermTM SIT (EPI-200).