Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Phototoxicity in vitro

Currently viewing:

Administrative data

Link to relevant study record(s)

Adequacy of study:
key study
Study period:
28 Jan - 19 Feb 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Type of study:
in vitro 3T3 NRU phototoxicity test
according to guideline
OECD TG 432 (In Vitro 3T3 NRU Phototoxicity Test)
Version / remarks:
adopted 2019
Morphology of the cells was not recorded 24 h after incubation
according to guideline
OECD TG 432 (In Vitro 3T3 NRU Phototoxicity Test)
Version / remarks:
adopted 2004
according to guideline
EU Method B.41 ( In vitro 3T3 NRU Phototoxicity Test)
Version / remarks:
adopted 2008
GLP compliance:
yes (incl. QA statement)
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species / strain / cell type:
BALB/c 3T3
Negative solvent / vehicle controls:
Solvent control for test item: Earle's Balanced Salt Solution, EBSS, containing 1% (v/v) DMSO Solvent control for positive control: EBSS
Positive controls:
Positive control substance:
chloropromazine HCL
Details on test system and experimental conditions:

- type and composition of culture medium: Dulbecco’s Minimal Essential Medium (DMEM), supplemented with 10% NCS
- incubation conditions (CO2 concentration, temperature, humidity): 7.5 ± 0.5% CO2, 37 ± 1.5 °C
- seeding density: 1.9 - 2 x 10^4 cells per well were seeded in 100 μL culture medium
- duration of incubation (pre- and post-treatment): pre-incubation: 24 - 25 h, after treatment cells were washed with EBSS and incubated in the dark overnight

- concentrations used: 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 µg/mL (for both range finding and main experiment)
- rationale for selection of concentrations of the test chemical used in the presence and in the absence of irradiation: in both experiments (range finding and main) 1000 µg/mL was used in the absence and presence of irradiation as the highest concentration in accordance to the OECD Guideline. At higher concentrations often false positive results are produced.
- type and composition of treatment medium (buffered salt solution): EBSS medium, composition not reported
- duration of the chemical treatment: 1h in the dark, then 50 min in the dark or under radiation

- rationale for selection of the light source used: not reported
- manufacturer and type of light source and radiometer: Dr. Hönle Sol 500 solar simulator
- spectral irradiance characteristics of the light source: not reported
- transmission and absorption characteristics of the filter(s) used: filter H1 was used to keep the UVB irradiation as low as possible, so that the 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: 1.68
- duration of the UV/vis light exposure: 50 min
- UVA dose (irradiance x time), expressed in J/cm2: ~5
- temperature of cell cultures during irradiation and cell cultures concurrently kept in the dark: 26 °C

- composition of Neutral Red treatment medium: serum free medium containing 50 µg/mL Neutral Red
- duration of Neutral Red incubation: 3 h
- incubation conditions (CO2 concentration; temperature; humidity): 7.5 ± 0.5% CO2, 37 ± 1.5 °C
- Neutral Red extraction conditions (extractant, duration): medium was removed completely and 0.15 mL of a solution of 49% (v/v) deionised water, 50% (v/v) ethanol and 1% (v/v) acetic acid was added , incubation: 10 min, room temperature
- wavelength used for spectrophotometric reading of Neutral Red optical density: 540 nm
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent: solubility of the test item
- Solubility of the test chemical in solvent: not reported but no precipitation was mentioned
- Justification for percentage of solvent in treatment medium: not reported
Evaluation criteria:
Evaluation criteria:
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

The following criteria must be met to consider the test acceptable:
After irradiation with a UVA dose of ~ 5 J/cm² the cell viability of the solvent control is > 80% of non-irradiated cells.
For the positive control CPZ the factor (PIF) between the two IC50 values is > 6
The mean OD540 of solvent controls is > 0.4
No statistics were reported
Key result

- cell viability obtained at each concentration of the test chemical, expressed in percent viability of mean, concurrent solvent controls: cell viability ranged from 98.21 - 103.33% (range finding experiment) and 93.68-106.46% (main experiment) without irradiation and from 95.57 - 99.17 and 94.68 - 100.53% (main experiment) with irradiation

Therefore, no concentration response curve or IC50-values or a PIF could not be calculated. The resulting MPE values were -0.012 and -0.021.

- test acceptance criteria; concurrent solvent control: yes, cell viabilitycell viability of the solvent control was > 80% of non-irradiated cells (test item: 95.2% and 94.9%; positive control: 98.2%
and 95.0), the mean OD540 of solvent controls were > 0.4 (range: 0.8519 to 0.9959)

- absolute viability (optical density of Neutral Red extract) of irradiated and non-irradiated cells: solvent controlmeand were with irradiation in the OD range from 0.8519 - 0.9460 and without 0.8860 - 0.9959
- historic negative and solvent control data; means and standard deviations:
OD irradiated cultures: 0.728 ± 0.177, range 0.338 - 1.287
OD non irradiated cultures: 0.771 ± 0.182, range 0.373 - 1.279
Data of 301 studies performed from March 2006 until August 2015

- 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) = 0.23 μg/mL
IC50 value (without artificial sunlight) = 10.22 μg/mL
PIF = 45.37
MPE = 0.520

Main Experiment:
IC50 value (with artificial sunlight) = 0.51 μg/mL
IC50 value (without artificial sunlight) = 14.63 μg/mL
PIF = 28.71
MPE = 0.553

- historic positive control chemical data: IC50 (+Irr) and IC50(-Irr) and PIF, MPE; means and standard deviations:
IC50 value (with artificial sunlight) = 0.47 ± 0.29 μg/mL, range: 0.07 - 1.65 µg/mL
IC50 value (without artificial sunlight) = 14.70 ± 0.5.54 μg/mL, range: 0.45 - 40.65 µg/mL
PIF = 45.21 ± 35.77, range 7.80 – 236.80
MPE = 0.592 ± 0.117, range 0.245 - 0.906
(Data of 301 studies performed from March 2006 until August 2015)

Summarizted Data can be found in Table 1 in Attachment 1.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: see Results
Validity criteria fulfilled:
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 1000 µg/mL in the BALB/c 3T3 cells clone 31.

Description of key information

Key, M-394980-01-2, 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

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.

In this study (M-558166-01-1), 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 concentration of the test substance with and without irradiation was 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 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 1.9 - 2 x 10^4 cells per well in 100 μL culture medium 24 h before treatment. Incubation was 1h in the dark, followed by 50 min either in the dark or under irradiation (1.68 mW/cm2, ~5 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 45.37 and 28.71, MPE of 0.52 and 0.533 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.