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Diss Factsheets

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Feb 2018 to 23 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(4-bromo-3-formylphenoxy)benzonitrile
EC Number:
822-547-1
Cas Number:
906673-54-9
Molecular formula:
C14H8BrNO2
IUPAC Name:
4-(4-bromo-3-formylphenoxy)benzonitrile
Test material form:
solid: particulate/powder
Details on test material:
Off-white powder
Specific details on test material used for the study:
Purity/Composition: 98.7% Test item storage: At room temperature protected from light Stable under storage conditions until: 20 November 2019 (retest date) (taken from label)

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:
EpiDerm Skin Model (EPI-200, Lot no.: 27958, kit F and G,

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

Test for the Interference of the Test Item with the MTT Endpoint A test item may interfere with the MTT endpoint if it is colored 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 Color Interference by the Test Item PF-06932437 was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, at least 25 mg of the test item 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 color change was observed.

Test for Reduction of MTT by the Test Item PF-06932437 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 mg of the test item or 50 µL Milli-Q water as a negative control were 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. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.

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.

DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM, 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 41 - 80%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 – 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 Item Preparation The correction factor is not applicable for this test, therefore no correction was made for the purity/composition of the test item. The solid test item was applied directly on top of the skin tissue. PF-06932437 was spread to match the size of the tissue. To protect the test item from light amber colored glassware was used or glassware was wrapped in tin-foil.

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 was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue (see figure 1). The plates were incubated for approximately 1 hour at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before PF-06932437 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-06932437 and two for a 1-hour exposure. The skin was moistened with 25 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 25.0 to 31.7 mg of the solid test item was added into the 6-well plates on top of the skin tissues. For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

Cell Viability Measurement The DMEM 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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Skin tissue was moistened with 25 µL of Milli-Q water and at least 25 mg of PF-06932437 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

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure with test item
Value:
ca. 99
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure with test item
Value:
ca. 98
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no 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%. All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, PF-06932437 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate PF-06932437 for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)).  The possible corrosive potential of PF-06932437 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. Groton batch GR12302 of PF-06932437 was an off-white powder.  Skin tissue was moistened with 25 µL of Milli-Q water and at least 25 mg of PF-06932437 was applied directly on top of the skin tissue. The positive control had a mean relative tissue viability of 7.9% after the 1-hour exposure.  The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit 2.8) and the laboratory historical control data range.  In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was  10%, indicating that the test system functioned properly. 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 the test item compared to the negative control tissues was 99% and 98%, respectively.  Because the mean relative tissue viability for PF-06932437 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment PF-06932437 is considered to be not corrosive. In conclusion, PF-06932437 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.