Pirimiphos-methyl

Phototoxicity in vitro

Administrative data

Endpoint:

phototoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Date of solubility test: 28 April 2015, Study initiation: 06 May 2015, Experimental start: 20 May 2015, Experimental end: 22 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Type of study:

in vitro 3T3 NRU phototoxicity test

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Test material form:

liquid

Test system

Species / strain

Species / strain / cell type:

BALB/c 3T3

Details on mammalian cell type (if applicable):

Mouse fibroblast cell line, clone A31

Controls

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO (Applichem, Lot 1V00468) in Earle's balanced salt solution (EBSS, Gibco, Lot 1661075)

Positive controls:

yes

Positive control substance:

other: chlorpromazine

Remarks:

2-chloro-10-[3-dimethylaminopropyl] phenothiazine, Fluka, Lot SZBB208XV

Details on test system and experimental conditions:

Culture medium: Dulbecco’s Modified Eagle Medium (DMEM, ATCC / Lot: 62594674 ) with 4.5 g/L D-glucose. The medium was supplemented with the following items: 10% Calf Serum (CS; ATCC/ Lot: 62262399) and 1% Penicillin/Streptomycin (final concentration: 100 IU/100 μg/mL; Gibco / Lot: 1655310)
Neutral red stock solution: 0.4 g neutral red (Applichem; Lot 4E010665) and 100 mL H2O
Neutral red medium: 1 mL NR stock solution, 79 mL cell culture medium without calf serum
Neutral red desorb: 1% acetic acid, glacial, 50% ethanol, 49% water

Cells: The test was carried out with BALB/c 3T3 cells (ATCC, CCL-163, clone A31). Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and sub-cultured at least once before they were used in the in vitro 3T3 NRU phototoxicity test. Cells at passage number 83 were used. Cells were precultured in 75 cm2 culture flasks in DMEM with 10% calf serum at 37 ± 1°C and 5% CO2.

UVA sensitivity of cells: 6 microtiter plates with cells were irradiated with the UVA-doses 0 (dark control), 3, 5, 7, 9 and 11 J/cm2.

Dose groups:
Negative control - 1% DMSO in EBSS
Positive controls -UVA: chlorpromazine at 100, 31.6, 10.0, 3.16, 1.00, 0.316, 0.100 and 0.0316 μg/mL
Positive controls +UVA: chlorpromazine at 10, 3.16, 1.00, 0.316, 0.100, 0.0316, 0.0100, and 0.00316 μg/mL
Test substance: 50.00, 15.81, 5.00, 1.581, 0.500, 0.158, 0.050 and 0.016 μg/mL
Blank: EBSS

UV irradiation: For the phototoxicity test the solar simulator SOL-500 equipped with a H1-filter (Dr. Hönle) was used. The light source was filtered by the filter H1 (315 to 400 nm) to attenuate the highly toxic UVB wavelength (280 to 315 nm). The UVA irradiance was measured by a UVA meter with a spectral sensitivity in a range from 315 to 400 nm and a measuring range between 0 – 199.9 mW/cm2. The UVA radiometer (Dr. Hönle) was calibrated by the supplier using a calibrated UV meter.

Experimental procedure: A cell suspension of 1E05 cells/mL in culture medium was prepared. 100 μL culture medium were dispensed into the peripheral wells of a 96-well tissue culture microtiter plate (= blanks). In the remaining wells, 100 μL of a cell suspension of 1E05 cells/mL (= 1E04 cells/well) were dispensed. For each test item two plates were prepared: one for determination of cytotoxicity (- UVA), and the other for determination of photocytotoxicity (+ UVA).
The cells were incubated for 24 ± 2 h (5% CO2, 37 ± 1°C) until they formed a half-confluent monolayer. This incubation period allowed for cell recovery and adherence, and for exponential growth.
After incubation, cells were washed with 150 μL EBSS per well. The solutions of the test item and the positive control were diluted seven times at a ratio of √10. The positive control was tested in a full scale phototoxicity test on two plates in parallel to the test item. 8 different concentrations were applied to 6 parallel cultures each. 100 μL of the appropriate concentration of test item or just solvent (= negative control, rows 2 and 11) were added to the cells. The cells were then incubated in the dark for 60 minutes (5% CO2 , 37 ± 1°C).
To perform the (+ UVA) part of the assay, the cells were irradiated for 50 min through the lid of the 96-well plate with 1.5 - 1.7 mW/cm2 UVA (= 4.5 - 5.1 J/cm2). The positions of the plates were exchanged halfway through the irradiation (25 min.). The distance light source - test system was 64 cm. The temperature during irradiation was 26.4°C (+ UVA) and 23.6°C (- UVA). Duplicate plates (- UVA) were kept at room temperature in a dark box for 50 min (= - UVA exposure time).
After exposition cells were washed with 150 μL EBSS. EBSS was replaced with culture
medium and the plates were incubated (5% CO2, 37 ± 1°C) overnight (18 - 22 h).
Following the incubation the cells were washed with 150 μL EBSS. 100 μL neutral red (NR) medium were added and the plates were incubated at 37 ± 1°C, in a humidified atmosphere of 5% CO2, for 3 h.
After incubation, the NR medium was removed, and the cells were washed with 150 μL
EBSS. 150 μL NR desorb solution (freshly prepared ethanol/acetic acid) were added. The microtiter plate was shaken rapidly on a microtiter plate shaker for 10 min, until the NR had been extracted from the cells and had formed a homogeneous solution. Then the optical density of the NR extract was measured at 540 nm in a micro plate auto reader, using blanks as a reference.

Vehicle:

yes

Vehicle / solvent:

Solvent: dimethylsulphoxide (DMSO)
Dilutent: Earle's balanced salt solution (EBSS)

Evaluation criteria:

Relative cell viability, expressed as percentage of untreated controls, was calculated for each of the eight test concentrations. To predict the phototoxic potential, the concentration responses obtained in the presence (+ UVA) and in the absence (- UVA) of irradiation were compared at the EC50 level, i.e. at the concentration inhibiting cell viability by 50% in comparison with untreated controls.

If both, EC50 (- UVA) and EC50 (+UVA) cannot be calculated due to the fact that a test item does not show any cytotoxicity up to the highest concentration, this indicates no phototoxic potential.
If a test item is only cytotoxic + UVA and is not cytotoxic when tested - UVA, the PIF cannot be calculated, although this result indicates a phototoxic potential of
the test item. In such cases the mean photo effect (MPE) is analysed. The MPE is based on comparison of the complete concentration response curves and is defined as the weighted average across a representative set of photo effect values.

Interpretation of results:
- MPE < 0.1: “no phototoxicity”
- MPE > 0.1 and < 0.15: “probable phototoxicity”
- MPE > 0.15: “phototoxicity”

The test meets the quality criteria if
- negative controls in the + UVA experiment show a viability of not less than 80% of that of non-irradiated cells in the same solvent of the concurrent dark experiment (-UVA).
- the mean OD540 NRU of untreated controls is ≥ 0.4.
- the EC50 value of the positive control in the + UVA experiment is in the range of 0.1 - 2.0 μg/mL and in the - UVA experiment 7.0 - 90.0 μg/mL and the photo irritation factor (PIF) is at least 6.

Results and discussion

Results:

No EC50 value for the - UVA experiment is available, since the substance showed only slight cytotoxic effect up to the highest concentration. With the highest concentration of the substance in the non-irradiated part of the experiment (50 μg/mL), viability of the cells was reduced to 65.6% relative to the untreated negative controls. For the highest concentration of the substance in the irradiated part of the experiment (50 μg/mL), viability of the cells was reduced to 22.1% relative to the untreated negative controls. The EC50-value of the substance in the + UVA experiment was calculated:
EC50 + UVA: 30.85 μg/mL.
The photo-irritation-factor (PIF) could not be calculated since the substance showed no cytotoxic effects. Therefore, the mean photo effect (MPE) was calculated:
MPE = -0.004
Any MPE < 0.10 indicates no phototoxicity, the substance is therefore considered as
“not phototoxic“ in the BALB/c 3T3 NRU phototoxicity assay.

Results with reference substance (positive control):

The positive control showed cytotoxic and phototoxic effects. With the highest concentration of the positive control in the non-irradiated part of the experiment
(100 μg/mL), viability of the cells was reduced to -0.3% relative to the untreated negative controls. The EC50 value was calculated to 7.49 μg/mL. In the irradiated part of the experiment (highest test item concentration: 10 μg/mL), the EC50 value was calculated to 1.08 μg/mL

Any other information on results incl. tables

Quality criteria

Concurrent negative control: The mean OD540 of the untreated control was ≥0.4. The negative controls of the concurrent +UVA experiment showed a viability of ≥80% (91.61%) relative to the untreated controls (- UVA).

Concurrent positive controls: The EC50 values of the positive controls in the experiments with and without UVA-irradiation were determinedby linear regression.

EC50 +UVA: 1.08 μg/mL

EC50 - UVA: 7.49 μg/mL

PIF = 6.9, indicating a phototoxic effect

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the given conditions, the test substance showed a cytotoxic effect, but no phototoxic effect was observed.

Executive summary:

The phototoxicity potential of the substance was studied under GLP in an in vitro 3T3 NRU assay according to OECD technical guidance 432. The substance was dissolved in dimethylsulphoxide (DMSO) and diluted in a 1:100 ratio in Earle's balanced salt solution. BALB/c 3T3 cells were treated for one hour with different concentrations of the test substance dissolved in DMSO at 37 ± 1°C and further over a period of 50 min in absence and in presence of a non-cytotoxic dose of UVA light, respectively. One day after treatment, cytotoxicity was analysed as a measure of reduction of neutral red uptake and compared to the controls.
Under the given conditions, no EC50 value for the - UVA experiment with the substance could be determined, since it showed only slight cytotoxic effects up to the highest concentration of 50 μg/mL. Therefore, the PIF could not be calculated. The EC50 value for the + UVA experiment with the substance could be determined. Therefore, the mean photo effect (MPE) was calculated:
EC50 + UVA: 30.85 μg/mL.
MPE = -0.004
The MPE value <0.1 obtained in the experiment indicates that the substance was 'not phototoxic' under the conditions of the test. The quality criteria demonstrating a valid study were all met.