Ziram

Phototoxicity in vitro

Link to relevant study record(s)

Reference

Endpoint:

phototoxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jul - 19 Sep 2013

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:

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

Version / remarks:

adopted in 2004

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted in 2019

Deviations:

yes

Remarks:

cells not checked for mycoplasma,

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Qualifier:

according to guideline

Guideline:

other: Committee for Proprietary Medicinal Products (CPMP) Note for Guidance on Photosafety testing, EMEA, CPMP/SWP/398/01

Version / remarks:

adopted in 2002

GLP compliance:

yes

Species / strain / cell type:

BALB/c 3T3

Details on mammalian cell type (if applicable):

CELLS USED
-Type and source of cells: BALB/c 3T3 c31 cell line (ZEBET, Berlin, Germany)

Negative solvent / vehicle controls:

yes

Remarks:

Earle's Balanced Salt Solution (EBSS) containing 1% dimethyl sulfoxide (DMSO) in the absence and presence of irradiation

Positive controls:

yes

Positive control substance:

chloropromazine HCL

Remarks:

Chlorpromazine dissolved in EBSS: 6.25, 12.5, 25, 37.5, 50, 75, 100 and 200 µg/mL in the absence of irradiation 0.125, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0 and 4.0 µg/mL in the presence of irradiation

Details on test system and experimental conditions:

IN VITRO 3T3 NRU PHOTOTOXICITY TEST

INCUBATION BEFORE AND AFTER TREATMENT
- type and composition of culture medium: Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10% newborn calf serum (NCS)
- incubation conditions (CO2 concentration, temperature, humidity): The cell cultures were incubated at 37 ± 1.5 °C in a 7.5 ± 0.5% carbon dioxide atmosphere.
- duration of incubation (pre- and post-treatment): Approximately 24 - 26 h after seeding the cultures were treated with the test item. The plates with the test substance were pre-incubated in the dark for 1 h. Afterwards, one plate was irradiated for 50 ± 2 min, while the other plate was stored in the dark.

TREATMENT WITH THE CHEMICAL
- rationale for selection of concentrations of the test chemical used in the presence and in the absence of irradiation:
The highest applied concentrations of the test item was based on a range finding experiment with 31.25 µg/mL being the highest concentration used due to limited solubility of the test item.
- duration of the chemical treatment: for 50 ± 2 min and after removing of the test substance, cells were washed and further incubated overnight in fresh culture medium at 37 ± 1.5 °C and 7.5 ± 0.5% C02.

IRRADIATION
- manufacturer and type of light source and radiometer: Dr. Hönle Sol 500 solar simulator with a H1 filter used to keep the UVB irradiation as low as possible
- spectral irradiance characteristics of the light source: >320 nm
- characteristics of the radiometer and details on its calibration: Due to the inhomogeneous distribution of irradiation intensity the UVA intensity was measured at the complete area with a UV-meter. The homogeneous area was marked and the cultures were irradiated in this area. The solar simulator was switched on about 30 min prior to the start of experiment.
- UVA irradiance at this distance, expressed in mW/cm2: 2.55 - 2.7
- duration of the UV/vis light exposure: 50 ± 2 min
- UVA dose (irradiance x time), expressed in J/cm2: 7.65 - 8.1
- temperature of cell cultures during irradiation and cell cultures concurrently kept in the dark: with and without irradiation: 50 ± 2 min at 20 - 30 °C

NEUTRAL RED VIABILITY TEST
- composition of Neutral Red treatment medium: serum free medium containing 50 µg Neutral Red/mL
- duration of Neutral Red incubation: 3 h
- Neutral Red extraction conditions (extractant, duration):
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 additional approximately 10 min at room temperature and a brief agitation, the plates were transferred to a microplate reader.
- wavelength used for spectrophotometric reading of Neutral Red optical density: 540 nm

Vehicle:

yes

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: EBSS containing 1% DMSO

Evaluation criteria:

The effective dose where only 50% of the cells survived (ED50), the Photo-Irritancy-Factor (PIF), as well as the Mean Phototoxic Effect (MPE) were calculated.

Evaluation of Results
A Photo-irritation Factor (PIF) was calculated:
PIF = (ED50 in the absence of UV)/(ED50 in the presence of UV)
If a chemical was only cytotoxic after exposure to UV light, but was not cytotoxic when tested in its absence, the PIF could not be accurately calculated. In this situation, the phototoxic potential of the test article was demonstrated from determination of the Mean Photo Effect (MPE). The MPE is based on comparison of the complete concentration response curves. It is defined as the weighted average across a representative set of photo effect values.

Based on the results obtained, the test item is evaluated as follows:
- if PIF (Photo-Irritancy-Factor) < 2 or MPE (Mean Phototoxic Effect) < 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.

Acceptability of the Assay
The assay meets the acceptance criteria:
- if after irradiation with a UVA doseof 5 J/cm² the cell viability of the solvent control is > 80% of non irradiated cells.
- if for the positive control CPZ the factor (PIF) between the two ED50 valuesis > 6.
- if the mean OD540 of solvent controls is > 0.4.

Statistics:

No statistics were performed.

Results:

OSMOLALITY AND PH
The osmolarity and the pH value of the highest concentrated test item solution of each experiment were measured prior to the experiments. An influence of the test item on pH or osmolarity was not observed in both experiments.

IN VITRO 3T3 NRU PHOTOTOXICITY TEST
Range finding experiment
A dose dependent cytotoxicity was observed after treatment of cells with the test substance in the presence and absence of irradiation with artificial sunlight. The ED50 value of the test item under irradiation was 0.7927 µg/mL and 0.9368 µg/mL in the absence of the artificial sunlight. The PIF of the test item was, 1.194 and the MPE values were calculated as -0.001. The PIF as well as the MPE value do not indicate any phototoxic potential.

Main experiment
The main experiment confirmed the cytotoxic effects observed in the range finding experiment. The ED50 value of the test item under irradiation was 0.9478 µg/mL and 1.068 µg/mL in the absence of artificial sunlight. The PIF of the test item was 1.127 and the MPE value were calculated as -0.004. Again, PIF as well as the MPE values do not indicate any phototoxic potential.

With regard to the acceptance criteria, the mean of solvent control values of the irradiated group versus the non irradiated group was 98.1 % (RFE) and 106.0 % (ME) for the test item and 91.3 % (RFE) and 84.6 % (ME) for the positive control plate.

The positive control chlorpromazine induced phototoxicity in the expected range after irradiation with artificial sunlight. The ED50 values were calculated in the absence (RFE: 15.02 µg/mL,
ME: 10.88 µg/mL) and the presence (RFE: 0.7592 µg/mL, ME: 0.4262 µg/mL) of irradiation. The resulting PIF was 19.790 (RFE) and 25.749 (ME). The resulting MPE was 0.583 (RFE) and 0.669 (ME).
For details on the results, please refer to attachment 2 and 3 under "Overall remarks, attachments"

Historical control data were provided an can be found in attachment 1 under "Overall remarks, attachments".

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: cell viability was below 50% from 0.98 µg/mL on with and without irradiadiation.

Validity criteria fulfilled:

yes

Conclusions:

The phototoxic potential of the present substance was assessed in the in vitro 3T3 NRU phototoxicity test according to OECD TG 432 and GLP. Under the conditions used, the test substance did not have any phototoxic effects on BALB/c 3T3cells.

Description of key information

Key, report number 1564000, BALB/c 3T3 NRU
no phototoxic effects observed

Key value for chemical safety assessment

Results:

no phototoxicity

Additional information

The following study (Report number: 1564000) was performed similar to OECD 432 and GLP to assess the phototoxic potential of the test substance. The test was performed using BALB/c 3T3cells. The experiment was performed twice, the first experiment served as a range finding experiment and the second as the main experiment. 31.25 µg/mL of the test item, dissolved in DMSO with a final concentration of 1% in Earle's Balanced Salt Solution (EBSS), were applied as the highest concentration in the first experiment. In the main experiment, the highest tested concentration of the test item solved in DMSO was chosen as 3.0 µg/mL under and without irradiation. One test group of cells treated with the test item was irradiated with artificial sunlight for 50 minutes with 2.55 - 2.7 mW/cm² UVA, resulting in an irradiation dose of 7.65 - 8.1 J/cm² UVA. Another test group of test item treated cells were kept in the dark for 50 min. A dose-dependent cytotoxicity was observed after treatment of cells with the test substance in the presence and absence of irradiation with artificial sunlight in both experiments. The photo-irritation Factor (PIF) as well as the mean phototoxic effect (MPE) values do not indicate any phototoxic potential. The mean of solvent control values of the irradiated group versus the non-irradiated group met the acceptance criteria. The positive control chlorpromazine induced phototoxicity in the expected range.

In conclusion, the test substance is not considered to be phototoxic.