mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Specific investigations: other studies

Link to relevant study record(s)

Reference

Endpoint:

cytotoxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 July 2014 to 17 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

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:

2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of method:

in vitro

Endpoint addressed:

other: toxicity of the test material in vitro after irradiation with artificial sunlight

Species:

other: Mouse embryo cell line

Strain:

other: BALB/c 3T3 c31 cell line

Details on test animals or test system and environmental conditions:

Cell Cultures and Medium
Large stocks (Master Cell Stock) of the BALB/c 3T3 c31 cell line are stored in liquid nitrogen in the cell bank of the testing facility. The master cell stock has been characterised by the testing facility. A working cell stock is produced by multiplying from the master cell stock.
Thawed stock cultures were propagated at 37 ± 1.5 °C in 75 cm^2 plastic flasks. Seeding was done with about 1 × 10^6 cells per flask in 15 mL of Dulbecco’s Minimal Essential Medium (DMEM), supplemented with 10 % newborn calf serum (NCS). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 ± 1.5 °C in a 7.5 ± 0.5 % carbon dioxide atmosphere.

Route of administration:

other: In medium

Vehicle:

DMSO

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was dissolved in DMSO. The final concentration of the solvent in Earle's balanced salt solution (EBSS) was 1 % (v/v). The highest applied concentration of the test material was 1000 μg/mL in accordance with the OECD Guideline. At higher concentrations often false positive results are produced.

Analytical verification of doses or concentrations:

no

Dose / conc.:

7.81 other: µg/mL

Remarks:

With and without irradiation

Dose / conc.:

15.6 other: µg/mL

Remarks:

With and without irradiation

Dose / conc.:

31.3 other: µg/mL

Remarks:

With and without irradiation

Dose / conc.:

62.5 other: µg/mL

Remarks:

With and without irradiation

Dose / conc.:

125 other: µg/mL

Remarks:

With and without irradiation

Dose / conc.:

250 other: µg/mL

Remarks:

With and without irradiation

Dose / conc.:

500 other: µg/mL

Remarks:

With and without irradiation

Dose / conc.:

1 000 other: µg/mL

Remarks:

With and without irradiation

No. of animals per sex per dose:

2 x 10^4 cells per well were seeded in 100 μL culture medium. Each concentration of the test material was measured 6 times.

Control animals:

yes, concurrent vehicle

Details on study design:

- Solvent Controls
The solvent controls were each tested in 12 replicates. Solvent controls were performed as follows:
Cultures exposed to the solvent controls, stored in the dark during exposure.
Cultures exposed to the solvent controls, irradiated for 50 min during exposure.

- Solar Simulator
The irradiation was performed with a 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. 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 minutes prior to the start of the experiment. The absorption spectrum of the test material was determined in the range from 270 to 800 nm. The test material showed an absorption maximum between 271.0 and 290.0 nm.

- Treatment
Nearly 25 hours after seeding the cultures were treated with the test material. The treatment was performed according to the OECD guideline as follows:
1) The cultures were washed with EBSS.
2) 8 dilutions of the solved test material were tested on two 96-well plates (100 μL/well).
3) Both plates were pre-incubated for 1 hour in the dark.
4) After one hour, one 96-well plate was irradiated through the lid at 1.65 mW/cm^2 (~ 5 J/cm^2), for 50 ± 2 min at 25 °C, the other plate was stored for 50 ± 2 min at 25 °C in the dark.
5) After irradiation the test material was removed and both plates were washed twice with EBSS.
6) Fresh culture medium was added and the cells were incubated for approximately 21.5 hours at 37 ± 1.5 °C and 7.5 ± 0.5 % CO2.

Examinations:

Determination of Neutral Red Uptake
The medium was removed and 0.1 mL serum free medium containing 50 μg Neutral Red / mL were 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 additional approx. 10 min 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.

Positive control:

Chlorpromazine dissolved in EBSS was used as positive control. The following concentrations were applied:
Absence of irradiation: 6.25, 12.5, 25, 37.5, 50, 75, 100, 200 μg/mL chlorpromazine.
Presence of irradiation: 0.125, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 4.0 μg/mL chlorpromazine.
Each concentration of the positive control was measured 6 times.

Details on results:

Treatment of BALB/c 3T3 with the test material:
The ED50 values could not be determined, since the viability of the cells was not reduced with and without irradiation.
The PIF could not be determined, since no ED50 values could be calculated.
MPE = -0.004
Mean OD540 nm solvent control value (≙ viability) irradiated versus non-irradiated group: 94.3 %.

Treatment of BALB/c 3T3 with the Positive Control (chlorpromazine):
ED50 value (with artificial sunlight) = 0.98 μg/mL
ED50 value (without artificial sunlight) = 24.64 μg/mL
PIF = 25.14
MPE = 0.343
Mean OD540 nm solvent control value (≙ viability) irradiated versus non-irradiated group: 104.2 %.

Cytotoxic effects were not observed after treatment of cells with the test material, neither in the presence nor in the absence of irradiation with artificial sunlight. The cell viabilities were not relevantly reduced compared with the result of the solvent control after exposure of the different test material concentrations to the cells. Therefore, ED50-values or the PIF value could not be calculated. The MPE value was -0.004. Therefore, the test material is not phototoxic.

Historical Data of the Positive Control Chlorpromazine and the Solvent Control

	Positive Control					Solvent Control
	EC50 + UV [µg/mL]	EC50 - UV [µg/mL]	L+UV of L-UV	PIF	MPE	OD Irradiated Cultures	OD Non-Irradiated Cultures
Mean	0.48	14.74	94.32	44.73	0.595	0.707	0.754
Std. Dev.	± 0.28	± 5.58	± 7.62	± 35.78	± 0.116	± 0.171	± 0.178
Ranges	0.07 – 1.65	0.45 – 40.65	80.1 – 118.1	7.80 – 212.96	0.245 – 0.906	0.338 – 1.214	0.373 – 1.279

Data of 258 studies performed from April 2006 until March 2014

Conclusions:

Under the conditions of the study the test material did not have any phototoxic effects on BALB/c 3T3 cells.

Executive summary:

The phototoxic potential of the test material was assessed according to OECD Test Guideline 432 and EU Method B.41 and in compliance with GLP.

The test was performed using BALB/c 3T3 cells clone 31. 1000 μg/mL of the test material, dissolved in DMSO (final concentration of DMSO in EBSS: 1 % (v/v)), were applied as the highest concentration. The following concentrations of the test material were tested with and without irradiation: 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 μg/mL.

As solvent control EBSS containing 1 % (v/v) DMSO was used. Chlorpromazine was used as positive control. The following concentrations were applied: without irradiation 6.25, 12.5, 25, 37.5, 50, 75, 100, 200 μg/mL; with irradiation 0.125, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 4.0 μg/mL.

One test group of cells treated with the test material was irradiated with artificial sunlight for 50 minutes with 1.65 mW/cm^2 UVA, resulting in an irradiation dose of ~ 5 J/cm^2 UVA. Another test group of test material treated cells were kept in the dark for 50 minutes.

No cytotoxic effects were observed after treatment of cells with the test material, neither in the presence nor in the absence of irradiation with artificial sunlight. Therefore, ED50-values or a PIF value could not be calculated. The resulting MPE was -0.004. Consequently, the test material is classified as not phototoxic.

The acceptance criteria were met. The positive control chlorpromazine induced phototoxicity in the expected range after irradiation with artificial sunlight.

Under the conditions of the study the test material did not have any phototoxic effects on BALB/c 3T3 cells.

Description of key information

Under the conditions of the study the test material did not have any phototoxic effects on BALB/c 3T3 cells.

Additional information

The phototoxic potential of the test material was assessed according to OECD Test Guideline 432 and EU Method B.41 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test was performed using BALB/c 3T3 cells clone 31. 1 000 μg/mL of the test material, dissolved in DMSO (final concentration of DMSO in EBSS: 1 % (v/v)), were applied as the highest concentration. The following concentrations of the test material were tested with and without irradiation: 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1 000 μg/mL.

As solvent control EBSS containing 1 % (v/v) DMSO was used. Chlorpromazine was used as positive control. The following concentrations were applied: without irradiation 6.25, 12.5, 25, 37.5, 50, 75, 100, 200 μg/mL; with irradiation 0.125, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 4.0 μg/mL.

One test group of cells treated with the test material was irradiated with artificial sunlight for 50 minutes with 1.65 mW/cm^2 UVA, resulting in an irradiation dose of ~ 5 J/cm^2 UVA. Another test group of test material treated cells were kept in the dark for 50 minutes.

No cytotoxic effects were observed after treatment of cells with the test material, neither in the presence nor in the absence of irradiation with artificial sunlight. Therefore, ED50-values or a PIF value could not be calculated. The resulting MPE was -0.004. Consequently, the test material is classified as not phototoxic.

The acceptance criteria were met. The positive control chlorpromazine induced phototoxicity in the expected range after irradiation with artificial sunlight.

Under the conditions of the study the test material did not have any phototoxic effects on BALB/c 3T3 cells.