thiacloprid (ISO);(Z)-3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2-ylidenecyanamide;{(2Z)-3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide

Phototoxicity in vitro

Link to relevant study record(s)

Reference

Endpoint:

phototoxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 - 26 Feb 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Type of study:

in vitro 3T3 NRU phototoxicity test

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 2019

Deviations:

yes

Remarks:

Morphology of the cells was not recorded 24 h after incubation.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Hessisches 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: BALB/c 3T3, ZEBET, Berlin, Germany
- Absence of Mycoplasma contamination: not reported
- Cell passage number: not reported
- doubling time: 16-20 h
- Radiation sensitivity of cells from a particular passage range determined with the irradiation equipment used in the in vitro 3T3 NRU phototoxicity test: not reported

Negative solvent / vehicle controls:

yes

Remarks:

For test item: Earle's Balanced Salt Solution, EBSS, containing 1% DMSO For positive control: EBSS

Positive controls:

yes

Positive control substance:

chloropromazine HCL

Remarks:

Concentrations with artificial sunlight: 0.125, 0.250, 0.500, 0.750, 1, 2, 4 µg/mL, without artificial sunlight: 6.25, 12.50, 25.00, 37.50, 50.00, 75.00, 100.00, 200.00 µg/mL

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 (for culturing), for treatment: Earle's Balanced Salt Solution (EBSS)
- incubation conditions (CO2 concentration, temperature): 7.5 ± 0.5%, 37 ± 1.5 °C
- duration of incubation (pre- and post-treatment): 24 h between seeding and treatment, overnight incubation after treatment and washing

TREATMENT WITH THE CHEMICAL
- concentration of the test item with and without irradiation: 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500 µg/mL (the test was performed twice, the first experiment was a range finding experiment but concentrations were not changed because no toxicity was observed)
- rationale for selection of concentrations of the test chemical used in the presence and in the absence of irradiation: The highest concentration was in accordance with the OECD guideline, at higher concentrations, false positive results are likely to occur.
- type and composition of treatment medium (buffered salt solution): Earle's Balanced Salt Solution (EBSS)
- duration of the chemical treatment: 1 h in the dark, followed by 50 min of irradiation or continued incubation in the dark

IRRADIATION
- manufacturer and type of light source and radiometer: Dr. Hönle Sol 500 solar simulator with filter H1
- spectral irradiance characteristics of the light source: not reported
- transmission and absorption characteristics of the filter(s) used: produced wavelength was > 320 nm
- characteristics of the radiometer and details on its calibration: not reported
- distance of the light source from the test system: not reported
- UVA irradiance at this distance, expressed in mW/cm2: 1.65 mW/cm²
- duration of the UV/vis light exposure: 50 min
- UVA dose (irradiance x time), expressed in J/cm2: 4.95 J/cm²
- temperature of cell cultures during irradiation and cell cultures concurrently kept in the dark: 26 °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: 3h
- incubation conditions (CO2 concentration; temperature): 7.5 ± 0.5% CO2, 37 ± 1.5 °C
- Neutral Red extraction conditions (extractant, duration): 49:50:1 deionised water : ethanol : acetic acid for 10 min at room temperature, followed by brief agitation
- wavelength used for spectrophotometric reading of Neutral Red optical density: 540 nm

Vehicle:

yes

Vehicle / solvent:

- Solvent used: DMSO
- Solubility of the test chemical in solvent: yes
- Percentage of solvent in treatment medium: 1%

Evaluation criteria:

Accessibility
The assay meets the 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.

Evaluation
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

Statistics:

No statistics were reported.

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: cell viability ranged from 97.35 - 109.58% (range finding experiment) and 97.35-105.41% (main experiment) without irradiation and from 100.23 - 109.65% and 97.11-102.39% (main experiment) with irradiation.

Therefore, no concentration response curve or IC50-values or a PIF could not be calculated. The resulting MPE values were -0.003 and 0.015.

- test acceptance criteria; concurrent solvent control: yes, cell viability of the solvent control was > 80% of non-irradiated cells, the mean OD540 of solvent controls were > 0.4 (range: 0.8315-1.1467)
- absolute viability (optical density of Neutral Red extract) of irradiated and non-irradiated cells: solvent control means were with irradiation in the OD range from 0.8315-1.0006 and without 0.9081-1.1467)
- historic negative and solvent control data; means and standard deviations:
OD irradiated cultures: 0.707 ± 0.171, range 0.338 - 1.214
OD non irradiated cultures: 0.754 ± 0.178, range 0.373 - 1.279
Data of 204 studies performed from April 2006 until July 2013

- test acceptance criteria; concurrent positive control:
Range finding experiment:
ED50 value (with artificial sunlight) = 1.07 μg/mL
ED50 value (without artificial sunlight) = 12.18 μg/mL
PIF = 11.48
MPE = 0.396

Main Experiment:
IC50 value (with artificial sunlight) = 0.68 μg/mL
IC50 value (without artificial sunlight) = 12.28 μg/mL
PIF = 18.10
MPE = 0.368

- historic positive control chemical data: IC50 (+Irr) and IC50(-Irr) and PIF/MPE; means and standard deviations:
EC50 value (with artificial sunlight) = 0.46 ± 0.28 μg/mL, range: 0.07 - 1.65 µg/mL
EC50 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


Summarized results can be found in Attachment 1 (results dose-finding experiment) and 2 (results main experiment). Historical control data can be found in Attachment 3 in the attached background material.

Remarks on result:

no phototoxicity

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: see results

Reported statistics and error estimates:

Not applicable

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 at any concentration up to 500 µg/mL in the BALB/c 3T3 cells clone 31.

Description of key information

Key, M-480557-01-1, OECD 432, BALB/c 3T3 NRU phototoxicity test,
no cytotoxic effects with and without irradiation with artificial sunlight up to and including 500 µg/mL

Key value for chemical safety assessment

Results:

no phototoxicity

Additional information

A guideline study conducted under GLP conditions is available, which is considered suitable as key study for evaluation of the phototoxic potential of present substance.

The test substance was tested for phototoxicity using BALB/c 3T3 cells. Testing consisted of a range finder and a main experiment. In both experiments, the concentrations of the test substance with and without irradiation was 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL. The positive control was Chlorpromazine tested at concentrations of 6.25 – 200 µg/mL without radiation and 0.125 – 4.0 µg/mL with irradiation.

For the assay, cells were seeded in 96-well plates in a density of 2 x 10^4 cells per well in 100 μL culture medium 24 h before treatment. Incubation was 1 h in the dark, followed by 50 min either in the dark or under irradiation (1.65 mW/cm2, 4.95 J/cm2). After the irradiation period, cells were washed, fresh culture medium was added and they were incubated in the dark overnight in a cell incubator. For detection of neutral red, cells were lysed with a mixture of deionized water:ethanol:acetic acid (49:50:1) for 10 min followed by absorbance determination at 540 nm. The IC50 values, the Photo-Irritancy-Factor (PIF), as well as the Mean Phototoxic Effect (MPE), were calculated if possible.

No decrease in cell viability was detectable in any test substance concentration, neither with nor without irradiation in both experiments. Since no cytotoxic effects were observed, neither in the presence nor in the absence of irradiation with artificial sunlight, no IC50-, PIF- and MPE values could be calculated. The positive control showed a marked increase in cytotoxicity when irradiated. (PIF of 11.48 and 18.10, MPE of 0.396 and 0.368 for the range finding and main experiment, respectively). Therefore, the test substance is not considered to be phototoxic.

The test was performed in accordance with the OECD test guideline 432, under GLP conditions, and is therefore valid.

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