Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Remarks:

Liver enzyme induction in rat hepatocytes

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 Jun 2010 to 16 July 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was intended to investigate the ability of the test material to induce cytochrome P450 (CYP) 2B (CYP2B) activity, CYP3A activity and cell proliferation in isolated female Han Wistar (HsdHanTM) rat hepatocyte cultures. Primary monolayer cultures of hepatocytes were prepared in 25 cm^2 flasks, 96- and 6-well plastic tissue culture plates in Leibowitz CL15 medium. Hepatocytes were cultured in Leibowitz CL15 medium for 4 h before the medium was removed and replaced with medium containing either the test material (1, 3, 10, 30, 65 and 100 µM), Phenobarbital sodium salt (positive control) or vehicle alone (negative control, 0.5% v/v dimethyl sulfoxide). After 96 h in culture, hepatocytes were either fixed using methanol at -20 °C (for assessment of replicative DNA synthesis) or harvested (for assessment of PROD and BROD activities) by scraping them into SET buffer, sonicating the mixture and storing it at –70 °C until analysis. Protein in hepatocytes scraped into SET buffer was determined.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

carcinogenicity

Test material

Test material information

Constituent 1

Reference substance name:

632-619-2

EC Number:

632-619-2

Cas Number:

881685-58-1

Molecular formula:

C20 H23 F2 N3 O

IUPAC Name:

632-619-2

Administration / exposure

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 other: µM

Doses / concentrations 2

Dose / conc.:

3 other: µM

Doses / concentrations 3

Dose / conc.:

10 other: µM

Doses / concentrations 4

Dose / conc.:

30 other: µM

Doses / concentrations 5

Dose / conc.:

65 other: µM

Doses / concentrations 6

Dose / conc.:

100 other: µM

Results and discussion

Details on results:

The positive control compound phenobarbital sodium salt (PB) induced the expected effects (increased cell proliferation and induction of CYP2B) in cultured female rat hepatocytes, indicating that the experimental system responded as expected. The test substance induced similar effects, with increased cell proliferation and induction of CYP2B.

Any other information on results incl. tables

Adenosine 5’-triphosphate (ATP)

Treatment with 65 and 100 μM of the test material resulted in significant cytotoxicity, with intracellular ATP levels being reduced to 2 and 0.5 % of control, respectively. No concentration of PB tested induced toxicity. 

 

Pentoxyresorufin-O-depentylation (PROD) 

Treatment with 10 and 30 μM of the test material resulted in statistically significant increases in PROD activity to 6.6- and 4.1-fold control, respectively. Treatment with 100 and 1000 μM PB resulted in statistically significant increases in PROD activity to 4.0- and 5.8-fold control, respectively.

 

Benzyloxyresorufin-O-debenzylation (BROD)

Treatment with 3, 10 and 30 μM of the test material resulted in statistically significant increases in BROD activity to 1.4-, 2.2- and 2.7-fold control, respectively. Treatment with 100 and 1000 μM PB resulted in statistically significant increases in BROD activity to 2- and 2.6-fold control, respectively.

 

Replicative DNA synthesis (S-phase)

Treatment with 25 ng/mL EGF resulted in a statistically significant increase in replicative DNA synthesis to 4.4-fold control, indicating that the hepatocytes could proliferate following exposure to proliferative stimuli and therefore demonstrating their suitability for use in investigations involving assessing induction of proliferation. Treatment with the test material resulted in statistically significant increases in replicative DNA synthesis at all concentrations tested, to a maximum of 2.6-fold control at 3 μM. Treatment with PB resulted in concentration-dependent increases in replicative DNA synthesis, to a maximum of 2.4-fold control at 1000 μM.

 

Table 1. Biochemical parameters in cultured female rat hepatocytes.

Treatment	ATP luminescence units released^a	S-phase labelling index (%)^b	PROD pmol resorufin/min/mg^c	BROD pmol resorufin/min/mg^c
Vehicle control (0.5% v/v DMSO)	98723±3184 (100.0±3.2)	5.01±0.56 (100.0±11.1)	0.062±0.095 (100.0±153.8)	1.22±0.17 (100.0±13.6)
PB 10 µM	100406±1704 (101.7±1.7)	8.26±0.97** (164.9±19.3)	0.068±0.028 (109.8±45.8)	1.34±0.24 (110.4±19.4)
PB 100 µM	103556±4897 (104.9±5.0)	10.82±0.80** (215.9±16.1)	0.244±0.046* (395.5±73.7)	2.47±0.20** (202.8±16.2)
PB 1000 µM	111624±3676** (113.1±3.7)	12.16±0.36** (242.7±7.2)	0.361±0.042** (584.3±67.5)	3.18±0.60** (261.6±49.3)
Test Material 1 µM	104243±2998* (105.6±3.0)	9.63±0.59** (192.2±11.7)	0.091±0.060 (147.0±96.9)	1.64±0.82 (135.2±67.8)
Test Material 3 µM	105273±4010* (106.6±4.1)	12.90±1.04** (257.4±20.8)	0.179±0.060 (289.6±97.1)	1.66±0.17* (136.9±14.0)
Test Material 10 µM	104996±3428** (106.4±3.5)	11.34±1.12** (226.4±22.4)	0.410±0.118* (664.3±190.8)	2.69±0.35** (221.4±28.6)
Test Material      30 µM	95483±4674 (96.7±4.7)	10.94±2.19** (218.4±43.7)	0.252±0.012* (408.0±19.8)	3.33±0.69** (274.2±56.9)
Test Material 65 µM	1957±118** (2.0±0.1)	9.74±1.32^d (194.5±26.3)	0.177±0.046^d(286.0±74.7)	1.36±0.20^d (111.8±16.4)
Test Material 100 µM	464±46** (0.5±0.1)
EGF 25 ng/mL		22.11±1.79** (441.4±35.6)

Values are mean ±SD, values in parenthesis are mean % control ±SD.

^an = 6 per group,^bn = 5 per group,^cn = 3 per group.

*Statistically different from control (Student’s t-test (2-sided) p<0.05; ** p<0.01).

^dNo statistical analyses performed due to excessive cytotoxicity.

Applicant's summary and conclusion

Conclusions:

The test material increased cell proliferation and induction of CYP2B, which was similar to the effects of the positive control compound phenobarbital sodium salt (PB).

Executive summary:

This study investigated the ability of the test material to induce cytochrome P450 (CYP) 2B (CYP2B) activity, CYP3A activity and cell proliferation (measured as the change in replicative DNA synthesis (S-phase of the cell cycle)) in isolated female Han Wistar rat hepatocyte cultures. Phenobarbital sodium salt (PB) was included as a positive control. Primary monolayer cultures of hepatocytes were prepared in 25 cm^2 flasks, 96- and 6-well plastic tissue culture plates in Leibowitz CL15 medium. Hepatocytes were exposed to the test material at 6 concentrations (1, 3, 10, 30, 65 and 100 µM), PB at 3 concentrations (10, 100 and 1000 μM) or vehicle (0.5 % v/v dimethyl sulfoxide (DMSO)) alone for 96 h. There were 3 replicates for each concentration in 25 cm^2 flasks for CYP activity measurements (measured as pentoxyresorufin-O-depentylation (PROD) and benzyloxyresorufin-O-debenzylation (BROD) for CYP2B and CYP2B/3A, respectively), 5 replicates for each concentration in 6-well plates for replicative DNA synthesis (proliferation) analysis and 6 replicates for each concentration in 96-well plates for cell toxicity measurements (measured as the change in cellular adenosine-5’-triphosphate (ATP)).

Treatment with 65 and 100 μM of the test material resulted in significant cytotoxicity, with intracellular ATP levels being reduced to 2 and 0.5 % of control, respectively. As a result, CYP activity and replicative DNA synthesis measurements made following treatment with these concentrations were excluded from data analysis. No concentration of PB tested induced toxicity. Treatment with 10 and 30 μM of the test material resulted in statistically significant increases in PROD activity to 6.6- and 4.1-fold control, respectively. Treatment with 100 and 1000 μM PB resulted in statistically significant increases in PROD activity to 4.0- and 5.8-fold control, respectively. Treatment with 3, 10 and 30 μM of the test material resulted in statistically significant increases in BROD activity to 1.4-, 2.2- and 2.7-fold control, respectively. Treatment with 100 and 1000 μM PB resulted in statistically significant increases in BROD activity to 2- and 2.6-fold control, respectively. Treatment with the test material resulted in statistically significant increases in replicative DNA synthesis at all concentrations tested, to a maximum of 2.6-fold control at 3 μM. Treatment with PB resulted in concentration-dependent increases in replicative DNA synthesis, to a maximum of 2.4-fold control at 1000 μM.

The test material increased cell proliferation and induction of CYP2B, which was similar to the effects of the positive control compound phenobarbital sodium salt (PB).