Desmedipham

Phototoxicity in vitro

Link to relevant study record(s)

Reference

Endpoint:

phototoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2013-08-20 to 2013-11-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Type of study:

in vitro 3T3 NRU phototoxicity test

Qualifier:

according to guideline

Guideline:

EU Method B.41 ( In vitro 3T3 NRU Phototoxicity Test)

Deviations:

yes

Remarks:

Principles of method if other than guideline

Qualifier:

according to guideline

Guideline:

OECD TG 432 (In Vitro 3T3 NRU Phototoxicity Test)

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Qualifier:

according to guideline

Guideline:

other: Committee for Proprietary Medicinal Products (CPMP), CPMP/SWP/398/01

Deviations:

yes

Remarks:

Principles of method if other than guideline

Principles of method if other than guideline:

The study was performed according to OECD guideline 432 In Vitro 3T3 NRU Phototoxicity Test. It is unclear whether UV sensitivity and absence of mycoplasma is checked regularly. Unclear if intensity of light and spectrum was measured through the same type of 96-well plate lid as was used in the assay. 2*10^4 cells per well was used instead of the 1*10^4 cells per well recommended in the guideline. Impurity profile is not known.
The filter was >320 nm in the study and the absorption maximum is below that (stated as 272 to 289 nm in study report; this is in the range in which cytotoxicity is expected). This filter was used to keep UVB as low as possible. Molar extinction coefficient is above 10 mol-1cm-1 at 313-320 nm. Therefore, phototoxicity is not covered at these wavelengths, but molar extinction coefficient is only above 1000 mol-1cm-1 (Cichy M., Poerschke R., 1999, M-186720-01-1) at wave lengths below 313 nm.
The irradiation was performed at 2.25-2.85 mW/cm2 (6.75-8.55 J/cm2) for 50 ± 2 min at 20-30 °C instead of 1.5-1.9 mW/cm2 (J/cm2) for 50 ± 2 min as it is stated in the study plan. Since the acceptance criteria were met and the UV dose of 5 J/cm2 is only a suggestion and not a demand by the OECD guideline it can be stated that this deviation did not affect the outcome of the study.
Study is acceptable.

GLP compliance:

yes (incl. QA statement)

Details on test system and experimental conditions:

Cell Cultures and Medium: The BALB/c 3T3 cell line was isolated from the muscle tissue of the mouse embryo. This fibroblast cell line has a high proliferation rate and a high plating efficiency of untreated cells which is necessary for the appropriate performance of the study. Large stocks (Master Cell Stock) of the BALB/c 3T3 c31 cell line (supplied by Dr. Liebsch, ZEBET, Berlin, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR. The master cell stock has been characterised by Harlan CCR. A working cell stock is produced by multiplying from the master cell stock.
Positive control: Chlorpromazine
Solar Simulator: Dr. Hönle Sol 500 solar simulator. The filter H1 was used to keep the UVB irradiation as low as possible. The produced wavelength of the solar simulator with the filter was > 320 nm. The test item showed absorption maxima between 272.0 to 289.9 nm.
Seeding of the Cultures: 2 x 10^4 cells per well were seeded in 100 µL culture medium (two plates, one was exposed to artificial sunlight, one was kept in the dark).
Treatment: 24 hours after seeding the cultures were treated with the test item. The treatment was performed according to the OECD guideline as follows: the cultures were washed with EBSS, 8 dilutions of the solved test item were tested on two 96-well plates (100 µL/well) both plates were pre-incubated for 1 hour in the dark, after one hour one 96-well plate was irradiated through the lid at 2.55 mW/cm2 (7.65 J/cm2), for 50 min at 27-28 °C, the other plate was stored for 50 min at 27-28 °C in the dark. After irradiation the test item was removed and both plates were washed twice with EBSS. fresh culture medium was added and the cells were incubated about 21 hours at 37 ± 1.5 °C and 7.5 ± 0.5% CO2.
Determination of cytotoxicity: The medium was removed and 0.1 mL serum free medium containing 50 µg Neutral Red / mL was added to each well. The plates were returned to the incubator for another 3 hours to allow uptake of the vital dye into the lysosomes of viable cells. Thereafter, the medium was removed completely and the cells were washed with EBSS. Then 0.15 mL of a solution of 49% (v/v) deionised water, 50% (v/v) ethanol and 1% (v/v) acetic acid were added to each well to extract the dye. After an additional approximately 10 minutes at room temperature and a brief agitation, the plates were transferred to a microplate reader (Versamax®, Molecular Devices) equipped with a 540 nm filter to determine the absorbance of the extracted dye. This absorbance showed a linear relationship with the number of surviving cells.
Number of measurements: Desmedipham and positive control: 6 times Solvent control: 12 times.

Vehicle:

yes

Vehicle / solvent:

DMSO (further diluted in EBSS, final concentration in EBSS was 1% (v/v).

Evaluation criteria:

Evaluation of results:
Based on the results obtained, the test item is evaluated as follows:
If PIF < 2 or MPE < 0.1: no phototoxic potential predicted.
If PIF > 2 and < 5 or MPE >0.1 and <0.15 a probable phototoxic potential is predicted.
If PIF > 5 or MPE > 0.15 a phototoxic potential predicted

Key result

Results:

The study was performed to assess the phototoxic potential of Desmedipham technical material. The test was performed using BALB/c 3T3 c31 cells clone 31. Two experiments were performed. The first experiment served as range finder (RFE), the second experiment (ME) was the confirming experiment.
62.5 µg/mL of the test item, dissolved in DMSO (final concentration of DMSO in EBSS: 1% (v/v)) was applied as the highest concentration in both experiments. The results were as shown in Table 1.
Cytotoxic effects did not occur after exposure of the test item to the cells, neither in the presence nor in the absence of irradiation with artificial sunlight in both experiments. Therefore, ED50-values or a PIF could not be calculated.

Remarks on result:

no phototoxicity

Validity criteria fulfilled:

yes

Conclusions:

Desmedipham is not phototoxic in this in vitro test system under the conditions of the study (wavelengths used was >320 nm as to keep UVB as low as possible).

Executive summary:

The phototoxic potential of desmedipham TC, was tested in this assay using BALB/c 3T3 cells clone 31. The experiment was performed twice. The first experiment served as a range finding experiment (RFE), the second one was the main experiment (ME). The following concentrations of the test item solved in DMSO (EBSS was 1% (v/v)); were tested in the presence and in the absence of irradiation in both experiments: 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.50 μg/mL. Chlorpromazine was used as positive control. Cytotoxic effects did not occur after exposure of the cells to the test item, neither in the presence nor in the absence of irradiation with artificial sunlight in both experiments so that ED50 values or a PIF could not be calculated. The resulting MPE value was -0.052 and 0.000, respectively. Desmedipham was therefore not phototoxic in this study.

Description of key information

Test method/  species	Result	Assessment	Reference
OECD 432 - Phototoxicity	Desmedipham was not phototoxic under the conditions of this study	Supporting study	Heppenheimer (2013)

Key value for chemical safety assessment

Results:

no phototoxicity