2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Specific investigations: other studies

Administrative data

Endpoint:

phototoxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 2013 to 29 August 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed according to OECD Guideline No. 432 and EURL ECVAM DB-ALM (INVITTOX) Protocol No. 78.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted April 13, 2004

Deviations:

yes

Remarks:

Duration of the second incubation (-/+UVA1): 24 min instead of 50 min

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM (INVITTOX) Protocol n ° 78

Version / remarks:

Updated December 2008

Deviations:

yes

Remarks:

Duration of the second incubation (-/+UVA1): 24 min instead of 50 min

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: phototoxicity

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

EC Number:

815-961-9

Cas Number:

1374760-95-8

Molecular formula:

C17H17N3O2S

IUPAC Name:

2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Test material form:

solid: particulate/powder

Details on test material:

- Physical state : White powder
- Storage condition of test material: Dry place, protected form light, (15 to 30°C)

Specific details on test material used for the study:

Molecular weight: 327.4
Storage conditions: room temperature, protected from light

Test animals

Species:

mouse

Strain:

Balb/c

Remarks:

Balb/c 3T3 mouse fibroblast cell line

Details on test animals or test system and environmental conditions:

TEST SYSTEM
The Balb/c 3T3 (mouse fibroblast) cell line was initially established in 1968 (Aaronson and Todaro) and is a very well characterized cell line.
BALB/c 3T3 (clone A31) cells were cultured (passage number < 100) in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 4 mM L-glutamine and 10% Newborn Calf Serum (NCS). Cells were seeded at a density of ~10,000 cells per well in 100 μL of growth media in 96-well sterile culture plates for all aspects of the study. The cells were assessed for UVA sensitivity per the ECVAM approved INVITTOX protocol (n ° 78). The cells were kept at ~37°C with ~5% CO2 in a humidified incubator (~24 hours) until ready to dose.

Administration / exposure

Vehicle:

DMSO

Remarks:

1% dimethyl sulfoxide

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared in DMSO at 100X final concentration. The test substance stock solutions were diluted in HBSS to 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL final concentrations. Chlorpromazine was also prepared at 100X in DMSO, and dissolved in HBSS for final concentrations of 0.0316, 0.1, 0.316, 1, 3.16, 10, 31.6, 100 μg/mL.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

1 hour exposure in dark + ca. 24 minutes (in presence or absence of UVA)

Frequency of treatment:

Once

Post exposure period:

Overnight (18-24 hours)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: µg/mL

Remarks:

Final concentration

Doses / concentrations 2

Dose / conc.:

0 other: µg/mL

Remarks:

Final concentration

Doses / concentrations 3

Dose / conc.:

0.001 other: µg/mL

Remarks:

Final concentration

Doses / concentrations 4

Dose / conc.:

0.01 other: µg/mL

Remarks:

Final concentration

Doses / concentrations 5

Dose / conc.:

0.1 other: µg/mL

Remarks:

Final concentration

Doses / concentrations 6

Dose / conc.:

1 other: µg/mL

Remarks:

Final concentration

Doses / concentrations 7

Dose / conc.:

10 other: µg/mL

Remarks:

Final concentration

Doses / concentrations 8

Dose / conc.:

100 other: µg/mL

Remarks:

Final concentration

No. of animals per sex per dose:

2 wells per dose (1 without UVA exposure and 1 with UVA exposure)

Details on study design:

EXPOSURE:
Test substance and chlorpromazine were prepared at 100X concentrations in 100% DMSO then diluted to exposure concentrations in HBSS.
Growth media was decanted and the cells gently washed with 150 μL HBSS. Duplicate plates were dosed with 100 μL of the diluted test articles, 100 µL of diluted negative control (1% DMSO) and 100 μL of the diluted chlorpromazine. The plates were placed back in the incubator for 1 hour before UVA exposure.
One chlorpromazine, one negative control (1% DMSO) and one test substance plate (-UVA) were placed in the dark at room temperature. One chlorpromazine, one negative control (1% DMSO) and one test substance plate (+UVA) were placed on a liquid-cooled shelf that is set ~56.5 cm from the UV light source (Honle UVASpot 400T). Cells designated +UVA were exposed to achieve a dosage of ~5 J/cm2 for approximately 24 minutes. At approximately half the time calculated for 5 J/cm2, the plates in the UVA were rotated 180° to ensure even exposure. After UVA exposure, all plates were decanted and the cells were washed twice with 150 μL HBSS followed by addition of 100 μL growth media. Plates were placed back into a ~37°C, ~5% CO2 humidified incubator overnight (18-24 hours) followed by NRU analysis.

NEUTRAL RED UPTAKE (NRU) ASSAY:
Neutral Red (NR) solution (3.3 mg/mL) was prepared at a final concentration of 50 μg/mL in serum free BALB/c 3T3 growth media. Growth media was decanted and the cells were washed once with 150 μL HBSS. One hundred microliters NR solution was added to each well and the plates were placed in the incubator for ~3 hours. Just prior to use, NR desorb solution was prepared by mixing 50 parts of 200 proof ethanol, 49 parts ultra-pure H2O and 1 part Acetic Acid. Plates were removed from the incubator and cells were gently washed with 150 μL HBSS, followed by addition of 150 μL NR desorb solution. Plates were placed on a DPC MicroMix 5 plate shaker at room temperature for ~10 minutes. Absorbance was determined at ~535 nm using a Biotek Synergy H4 or equivalent plate reader. Absorbance values for both vehicle controls (-UVA and +UVA) were determined and an average percent viability was calculated for each sample. The results of each condition were evaluated and compared to vehicle controls. Data were graphed and the EC50 and PIF (Photo-Irritation Factor) were generated for each test article, if applicable.

Results and discussion

Details on results:

The assay met all the critical acceptance criteria as outlined in INVITTOX Protocol n ° 78. The test material did not reduce cell viability below 68%. Therefore, no EC50 could be calculated resulting in a PIF of *1 predicting no phototoxic potential.

Any other information on results incl. tables

Assay acceptance criteria

Assessment	Criteria	Study result
Balb/c 3T3 UVA Sensitivity Check	Viability of cells exposed to 5 J/cm2 is at least 80% of non-irradiated cells	84.4 %
Balb/c 3T3 UVA Sensitivity Check	Viability of cells exposed to 9 J/cm2 is at least 50% of non-irradiated cells	50.0 %
Assay Quality Check	Absolute OD540 of untreated controls is > 0.3	Range of 0.373 - 0.460
Assay Quality Check	The left and right vehicle controls do not differ more than 15% from mean of all controls	Highest deviation is 5.5 %
Positive Control Check	EC50 of chlorpromazine exposed cells + UVA is in the range of 0.1 μg/mL to 2.0 μg/mL	0.21
Positive Control Check	EC50 of chlorpromazine exposed cells - UVA is in the range of 7.0 μg/mL to 90.0 μg/mL	31.33
Positive Control Check	The PIF between the two EC50 values is at least 6	147.5

Detailled results are provided in attachment in section "Attached background material".

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test substance is not predicted to have phototoxic potential.

Executive summary:

The potential phototoxicity of the test material was assessed using the INVITTOX 3T3 NRU Phototoxicity Assay according to the OECD Guideline No. 432 and the EURL ECVAM DB-ALM (INVITTOX) Protocol No. 78. Cell viability was determined by Neutral Red Uptake (NRU) after exposing the tissues to 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of the test material in the presence and absence of 5 J/cm². After the exposures and an overnight recovery period, the NRU assay was performed and data were normalized to the vehicle control, 1% DMSO in culture media. The test material was exposed at concentrations up to and including the highest concentration allowable under the INVITTOX 3T3 NRU Phototoxicity (100 μg/mL).

The cell viability never dropped below 68% viability, both in the presence and absence of UVA. Therefore an EC50 could not be calculated in either condition resulting in a PIF ( (Photo-Irritation Factor) of *1, predicting no phototoxic potential.