Pyrimidine, 4-chloro-6-ethenyl-5-fluoro-

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2016 to 22 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-6-ethenyl-5-fluoropyrimidine

EC Number:

940-222-3

Cas Number:

1403893-61-7

Molecular formula:

C6 H4 Cl F N2

IUPAC Name:

4-chloro-6-ethenyl-5-fluoropyrimidine

Test material form:

liquid

Details on test material:

received as orange brown liquid

Specific details on test material used for the study:

Batch 2000533255 Purity/Composition 99.2% Test item storage In refrigerator (2-8°C) Stable under storage conditions until Not indicated

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm2) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
Rationale
Recommended test system in international guidelines (OECD and EC).
Source
MatTek Corporation, Ashland MA, U.S.A.

Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.
Freeze-killed tissues (EPI-200, Lot no.: 23610 kit M) Living epidermis was transferred to a freezer (<-15°C), thawed, and then again transferred to (<-15°C). The freeze-killed epidermis was stored at < -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 ml DMEM medium. Further use of killed tissues was similar to living tissues.
DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM medium, serum-free supplied by MatTek Corporation.
MTT medium
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
Environmental conditions
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 66 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 - 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test for the interference of the test item with the MTT endpoint
A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for colour interference by the test item

PF-06645243 was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 50 µl of
PF-06645243 or 50 µl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.

Test for reduction of MTT by the test item

PF-06645243 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µl of PF-06645243 was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently.
Since the test item reacts with the MTT medium in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed non treated tissues must be used for the cytotoxicity evaluation with MTT. At the end of the exposure time it was checked if a blue / purple colour change was observed.

Application/Treatment of the test item
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay is started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The level of the DMEM medium is just beneath the tissue (see fig 1). The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before PF-06645243 was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to PF-06645243 and two for a 1-hour exposure. Fifty µl of the undiluted test item was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 µl Milli-Q water (negative control) and with 50 µl 8N KOH (positive control), respectively. In addition for the 3 minute and 1 hour exposure two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Cell viability measurement
The DMEM medium was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

PF-06645243 was applied undiluted (50 µl) directly on top of the skin tissue. Skin tissue was moistened with 25 µl of Milli-Q water and at least 25 mg of PF-06645243 was applied directly on top of the skin tissue.

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure with test item

Value:

ca. 59

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure with test item

Value:

ca. 15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be <_ 30%.
d) The non-specific MTT reduction should be <_ 30% relative to the negative control OD.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

Finally, it is concluded that this test is valid and that PF-06645243 is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In vitro skin corrosion test with  PF-06645243 using a human skin model.

This report describes the ability of  PF-06645243 to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of

PF-06645243 was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 2000533255 of  PF-06645243 was a light yellow oil with a purity of 99.2%.

PF-06645243 was applied undiluted (50 µl) directly on top of the skin tissue. Skin tissue was moistened with 25 µl of Milli-Q water and at least 25 mg of  PF-06645243 was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 9% after the 1 hour exposure. The absolute mean OD570 (optical density at 570  nm)  of the negative control tissues was within the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 5%, indicating that the test system functioned properly.

PF-06645243 did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each timepoint. The non-specific reduction of MTT by

PF-06645243 was -1.1% and 3.4% of the negative control tissues after 3 minutes and 1 hour respectively. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues for the 1 hour treatment.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with

PF-06645243 compared to the negative control tissues was 59% and 5%, respectively. Because the mean relative tissue viability for  PF-06645243 was below 15% after the 1-hour treatment it is considered to be corrosive.

Finally, it is concluded that this test is valid and that  PF-06645243 is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.