Sodium 1-[6-(morpholin-4-yl)pyrimidin-4-yl]-4-(1H-1,2,3-triazol-1-yl)-1H-pyrazol-5-olate

Phototoxicity in vitro

Administrative data

Endpoint:

phototoxicity

Type of information:

experimental study

Adequacy of study:

other information

Study period:

12-01-2012 to 24-07-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

13 April 2004

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1-[6-(morpholin-4-yl)pyrimidin-4-yl]-4-(1H-1,2,3-triazol-1-yl)-1H-pyrazol-5-olate

EC Number:

875-892-5

Cas Number:

1375799-59-9

Molecular formula:

C13 H14 N8 O2 . Na

IUPAC Name:

Sodium 1-[6-(morpholin-4-yl)pyrimidin-4-yl]-4-(1H-1,2,3-triazol-1-yl)-1H-pyrazol-5-olate

Test system

Species / strain

Species / strain / cell type:

BALB/c 3T3

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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: DMEM (Dulbecco's Modified Eagle's Medium): No. 21885-025, Gibco (Eggenstein, Germany); FBS (Fetal Bovine Serum): No. S 1800, Biowest (Nuaille, France); NCS (Newborn Calf Serum): No. N-4637, Sigma (Deisenhofen); Penicillin/streptomycin solution: No. P-4333, 10000 U/mL Penicillin, 10 mg/mL
Streptomycin, Sigma (Deisenhofen)
- incubation conditions (CO2 concentration, temperature, humidity): The cells were cultivated in a humidified incubator at 37°C and 5% C02.
- duration of incubation (pre- and post-treatment): preincubation time 24h and postincubation time 24h

TREATMENT WITH THE CHEMICAL
- rationale for selection of concentrations of the test chemical used in the presence and in the absence of irradiation: according to OECD test guideline
- in case of limited solubility of the test chemical and absence of cytotoxicity: rationale for the highest concentration tested: The highest dose used for the test item corresponds to the solubility limit in PBS.
- type and composition of treatment medium (buffered salt solution): PBS
- duration of the chemical treatment: 1 h plus 50 min irradiation

IRRADIATION
- rationale for selection of the light source used: UV -lamp equipped with a H1 filter (SoB, Dr. Hönle GmbH, Gräfelfing) with an intensity of 1.7 mW/cm² (5 J/cm²) for 50 min

NEUTRAL RED VIABILITY TEST
- composition of Neutral Red treatment medium: 50 µL per well of a Neutral red stock solution (3.3 g/L) were added to the cell cultures for ca 2h at 37°C.

- incubation conditions (CO2 concentration; temperature; humidity): 37°C, 5%CO2
- Neutral Red extraction conditions (extractant, duration): 50% ethanol, 1% glacial acetic acid
- wavelength used for spectrophotometric reading of Neutral Red optical density: 540 nm, reference 630 nm

Vehicle:

yes

Vehicle / solvent:

DMSO

Evaluation criteria:

Phototoxicity determination

With the software "Phototox Version 2.0" (Federal Institute of Risk Assessment, Berlin), a Photo-Irritation-factor (PIF) was calculated directly from the raw data per single assay. PIF is defined as the ratio of the half effective concentration for cytotoxicity (EC50) without (-UV) and with UV-irradiation (+UV). In an iterative process, whereby datapoints were randomly excluded in each round, many - and +UV concentration response curves models were approximated, yielding mathematically determined EC50 values. Pairing each curve of the -UV experiment with each curve of the +UV experiment resulted in a set of PIFs. These were averaged and given as PIF Mean per assay, which is the output of the software. An overall average PIF was then be calculated from the 3 replicate assays. Based on the validation study (Spielmann et al. 1998), a test substance with a PIF ≤ 2 predicts "no phototoxicity", a PIF > 2 and< 5 predicts "probable phototoxicity", and a PIF ≥ 5 predicts "phototoxicity".

Results and discussion

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:
- concentration response curves (test chemical concentration vs. relative cell viability) obtained in concurrent +Irr and –Irr experiments:
- analysis of the concentration-response curves:
- computation/calculation of IC50 (+Irr) and IC50 (-Irr):
- 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:
In the present study the phototoxic potential of the test substance was investigated at the concentrations 0.1 µg/mL, 1 µg/mL, 3 µg/mL, 10 µg/mL, 30 µg/mL, 100 µg/mL 300 µg/mL, 500 µg/mL. Chlorpromazine hydrochloride (from 0.01- 100 µg/mL) was used as a positive control. The three independent assays with the test item yielded an average PIF = 1. Molidustat was thus identified as a non-phototoxic compound.

After treatment of the cells with chlorpromazine hydrochloride the average PIF was 27. Thus, as expected, chlorpromazine hydrochloride was identified as a phototoxic compound

For more detailed results please refer to 'Any other information on results incl. tables'

Any other information on results incl. tables

 

	PIF Mean per assay
Compound	Assay 1	Assay 2	Assay 3	Average PIF	Evaluation
Test item	1.119	0.923	1.055	1.032	non-phototoxic
Chlorpromazine-HCl	29.999	25.476	26.752	27.409	phototoxic

 

Applicant's summary and conclusion

Validity criteria fulfilled:

not applicable

Conclusions:

The three independent assays with Molidustat yielded an average PIF = 1, thus, it was identified as a non-phototoxic compound.

Executive summary:

In the present test conducted according to OECD test guideline 432, mouse fibroblasts were exposed to the test item in DMSO and to Chlorpromazine hydrochloride as positive control at concentrations of 0.1 µg/mL, 1 µg/mL, 3 µg/mL, 10 µg/mL, 30 µg/mL, 100 µg/mL 300 µg/mL, 500 µg/mL and Chlorpromazine hydrochloride (from 0.01- 100 µg/mL)), for 1h before radiation with a UV-lamp for 50 min. After a post-incubation period of 24h the viability was determined using neutral red. Each assay was performed in triplicate. Based on the validation study (Spielmann et al. 1998), a test substance with a PIF ≤ 2 predicts "no phototoxicity", a PIF > 2 and< 5 predicts "probable phototoxicity", and a PIF ≥ 5 predicts "phototoxicity", Molidustat was non-phototoxic under the conditions of the test.