clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine

Phototoxicity in vitro

Link to relevant study record(s)

Reference

Endpoint:

phototoxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 - 27 Nov 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 2004

Deviations:

yes

Remarks:

no microscopic evaluation of cell morphology and integrity of cell monolayer performed

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species / strain / cell type:

BALB/c 3T3

Details on mammalian cell type (if applicable):

CELLS USED
-Type and source of cells: fibroblast cell line isolated from the muscle tissue of the mouse embryo
- Absence of Mycoplasma contamination: Yes
- Cell passage number: < 100

Negative solvent / vehicle controls:

yes

Remarks:

solvent control for test item: Earle's Balanced Salt Solution, EBSS with 1% DMSO, solvent control for positive control: EBSS

Positive controls:

yes

Positive control substance:

chloropromazine HCL

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 supplemented with 10% NCS
- incubation conditions (CO2 concentration, temperature, humidity): 7 - 8%, 35.5 - 38.5 °C, not reported
- duration of incubation (pre- and post-treatment): 24 h prior incubation. After incubation, cells were washed with EBSS, fresh culture medium was added and cells were incubated over night under conditions mentioned above.

TREATMENT WITH THE CHEMICAL
- concentrations of test item used: 7.813, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL (range finding and main experiment)
- concentrations of positive control used: 6.25, 12.5, 25, 37.5, 50, 75, 100 and 200 µg/mL (without irradiation); 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2 and 4 µg/mL (with irradiation)
- rationale for selection of concentrations of the test chemical used in the presence and in the absence of irradiation: a range finding experiment was performed (first experiment)
- rationale for the highest concentration tested: the highest concentration tested was 1000 µg/mL as is in accordance with OECD TG 432, since at higher concentrations often false positive results are produced.
- type and composition of treatment medium (buffered salt solution): EBSS
- duration of the chemical treatment: 1 h in the dark, then 50 min in the irradiation machinery covered with or without tinfoil

IRRADIATION
- manufacturer and type of light source and radiometer: Dr. Hönle Sol 500 solar simulator
- transmission and absorption characteristics of the filter(s) used: H1 filter was used to keep the UVB irradiation as low as possible. The wavelength of the solar simulator with the filter was > 320 nm.
- characteristics of the radiometer and details on its calibration: the UV-meter is calibrated every year by an external service
- distance of the light source from the test system: 42 cm
- UVA irradiance at this distance, expressed in mW/cm2: 1.65
- duration of the UV/vis light exposure: 50 min
- UVA dose (irradiance x time), expressed in J/cm2: 4.95
- temperature of cell cultures during irradiation and cell cultures concurrently kept in the dark: 25 °C

NEUTRAL RED VIABILITY TEST
- composition of Neutral Red treatment medium: serum free medium with 50 µg/mL Neutral Red
- duration of Neutral Red incubation: 3 h
- incubation conditions (CO2 concentration; temperature; humidity): 7 - 8%, 35.5 - 38.5 °C, not reported
- Neutral Red extraction conditions (extractant, duration): deionised water/ethanol/acetic acid (49/50/1, v/v/v), 10 min
- wavelength used for spectrophotometric reading of Neutral Red optical density: 540 nm

Vehicle:

yes

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: the solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures
- Solubility of the test chemical in solvent: Yes, no precipitation at any concentration was observed after dissolving the test item and diluting the DMSO stock solution with EBSS

Evaluation criteria:

If PIF < 2 or MPE < 0.1, no phototoxic potential is 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 is predicted. Acceptability criteria: The assay meets acceptance criteria, if after irradiation with a UVA dose of 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 values is > 6. If the mean OD540 of solvent controls is > 0.4.

Statistics:

Not applicable

Key result

Results:

IN VITRO 3T3 NRU PHOTOTOXICITY TEST

- cell viability obtained at each concentration of the test chemical, expressed in percent viability of mean, concurrent solvent controls: 83.22 - 103.51% (range finding experiment; +Irr), 88.50 - 97.95% (range finding experiment; -Irr), 90.70 - 95.45% (main experiment; +Irr), 92.02 - 98.54% (main experiment; -Irr)
- concentration response curves (test chemical concentration vs. relative cell viability) obtained in concurrent +Irr and –Irr experiments: not applicable
- analysis of the concentration-response curves: not applicable
- computation/calculation of IC50 (+Irr) and IC50 (-Irr): not applicable
- comparison of the two concentration response curves obtained in the presence and in the absence of irradiation, either by calculation of the Photo-Inhibition-Factor (PIF), and/or by calculation of the Mean-Photo-Effect (MPE) depending on the dose-response curve: not applicable
- test acceptance criteria; concurrent solvent control:
- absolute viability (optical density of Neutral Red extract) of irradiated and non-irradiated cells: OD540nm ranged from 0.7637 - 0.9498 (range finding experiment; +Irr), 0.8663 - 0.9588 (range finding experiment; -Irr), 0.8951 - 0.9419 (main experiment; +Irr), and 0.9867 - 1.0566 (main experiment; -Irr).
- historic negative and solvent control data; means and standard deviations: OD540nm: 0.707 ± 0.171 (+Irr); 0.754 ± 0.178 (-Irr). Ranges: 0.338 - 1.214 (+Irr), 0.373 - 1.279 (-Irr) (data from 204 studies performed from April 2006 until July 2013).

- test acceptance criteria; concurrent positive control: Yes
- IC50(+Irr) and IC50(-Irr) and PIF/MPE of positive control chemical:
Range finding experiment:
IC50 value (with artificial sunlight) = 0.16 μg/mL
IC50 value (without artificial sunlight) = 8.94 μg/mL
PIF = 55.60
MPE = 0.705

Main Experiment:
IC50 value (with artificial sunlight) = 0.20 μg/mL
IC50 value (without artificial sunlight) = 16.58 μg/mL
PIF = 81.56
MPE = 0.644

- historic positive control chemical data: IC50 (+Irr) and IC50(-Irr) and PIF, MPE; means and standard deviations:
IC50 value (with artificial sunlight) = 0.46 ± 0.28 μg/mL, range: 0.07 - 1.65 µg/mL
IC50 value (without artificial sunlight) = 14.83 ± 5.87 μg/mL, range: 0.45 - 40.65 µg/mL
PIF = 45.49 ± 34.21, range 7.80 – 212.96
MPE = 0.595 ± 0.116, range 0.245 - 0.906
(Data of 204 studies performed from April 2006 until July 2013)

Remarks on result:

no phototoxicity

Results with reference substance (positive control):

- Results with reference substance valid? Yes

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. Under the conditions used, the test substance was not phototoxic in any concentration up to 1000 µg/mL in the BALB/c 3T3 cells clone 31.

Description of key information

Key, M-472655-02-1, OECD 432, BALB/c 3T3 NRU phototoxicity test,
no cytotoxic effects with and without irradiation with artificial sunlight up to and including 1000 µg/mL

Key value for chemical safety assessment

Results:

no phototoxicity

Additional information

One phototoxicity study is available (M-472655-02-1). This study was performed under GLP conditions and in accordance with OECD TG 432 with only minor deviations. It is therefore considered reliable and valid and suitable as a key study.

Testing consisted of a range finder and a main experiment. In both experiments the test item was administered to BALB/c 3T3 cells for 1 h prior to irradiation of cells with 4.95 J/cm² UVA (50 min at 1.65 mW/cm²) at concentrations of 7.813, 15.63, 31.25, 62.50, 125, 250, 500 and 1000 µg/mL. Another set of cells was treated in the same way, but was covered with tinfoil during irradiation (-Irr). EBSS with 1% DMSO served as control. As positive control chlorpromazine was used at concentrations of 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2 and 4 µg/mL (+Irr) and 6.25, 12.5, 25, 37.5, 50, 75, 100 and 200 µg/mL (-Irr). Determination of neutral red uptake served as a marker for cytotoxicity. 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-, PIF- and MPE values could not be calculated. The resulting MPE values were -0.021 and 0.026, respectively. In conclusion, it can be stated that under the experimental conditions tested here, the test item does not possess any phototoxic potential.