2-Pyridinecarboxamide, 4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]-3-fluorophenoxy]-N-methyl-, hydrochloride (1:1)

Bioaccumulation: aquatic / sediment

Administrative data

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2011 - Jan 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted with the base (BAY 73-4605) according to OECD guideline under GLP

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Deviations:

no

Principles of method if other than guideline:

not relevant

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-{4-[({[4-chloro-3-(trifluoromethyl)phenyl]amino}carbonyl)amino]-3-fluorophenoxy}-Nmethylpyridine- 2-[14C]carboxamide

IUPAC Name:

4-{4-[({[4-chloro-3-(trifluoromethyl)phenyl]amino}carbonyl)amino]-3-fluorophenoxy}-Nmethylpyridine- 2-[14C]carboxamide

Details on test material:

- Name of test material (as cited in study report): Regorafenib (14-C)
- Analytical purity: 98%
- Lot/batch No.: GCM 1569-1-13 A
- Radiochemical purity (if radiolabelling): 98%
- Specific activity (if radiolabelling): 98% / 2.55 MBq/mg

Radiolabelling:

yes

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

Lepomis macrochirus

Study design

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

14 d

Test conditions

Nominal and measured concentrations:

0.16 and 1.6 ¿g/L (nominal)
0.05 and 0.45 ¿g/L (measured)

Details on estimation of bioconcentration:

BASIS INFORMATION
- Measured/calculated logPow: 3.9

BASIS FOR CALCULATION OF BCF
- Estimation software: SAS 9.2
- Result based on measured log Pow of: 3.9

Results and discussion

Bioaccumulation factor

Bioaccumulation factor 1

Type:

BCF

Value:

2 874 dimensionless

Basis:

total lipid content

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:0.16

Bioaccumulation factor 2

Type:

BCF

Value:

4 444 dimensionless

Basis:

total lipid content

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:1.6

Depuration

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

6 d

Any other information on results incl. tables

TT 1:	Concentration of regorafenib in fish
Nominal concentration of regorafenib [ìg/L]	Accumulation phase	Depuration phase
Day 4 [µg/kg] (n=4)	Day 6 [µg/kg] (n=4)	Day 8 [µg/kg] (n=4)	Day 14 [µg/kg] (n=4)	Day 21 [µg/kg] (n=4)	Day 28 [µg/kg] (n=4)	Day 30 [µg/kg] (n=4)	Day 32 µg/kg] (n=4)	Day 38 [µg/kg] (n=4)	Day 42 [µg/kg]
0,16	59	78,6	93,9	149,2	143,8	138,2	102	51,9	9,8	8,8 (n=4)
1,6	789,9	1155,6	1498,4	2301,8	1690	2007,7	2042,2	1082,7	292	64,3 (n=2)
TT 2:	Concentration of regorafenib in water (exposure phase)
Nominal concentration of regorafenib [ìg/L]	Day 4 [ng/L]	Day 6 [ng/L]	Day 8 [ng/L]	Day 14 [ng/L]	Day 21 [ng/L]	Day 28 [ng/L]	Mean measured concentration (MV) [ng/L]
Control	0	0	0	0	0	0	0
0,16	71,5	54,4	15	39,3	28,6	69,6	46,4
1,6	722,6	426,6	314,7	348,7	207,1	702,8	453,8
TT 3:	Concentration of regorafenib in water (depuration phase)
Nominal concentration of regorafenib [ìg/L]	Day 30 [ng/L]	Day 32 [ng/L]	Day 38 [ng/L]	Day 42 [ng/L]	Mean measured concentration (MV) ng/L]
Control	0	0	0	0	0
0,16	15,8	7,4	11,9	25,5	15,2
1,6	76,9	25,5	16,3	12,8	32,8

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No substance related mortality or abnormal behavior was observed neither in the test nor in the vehicle control solutions during the whole
experimental period. Thus, it was shown that the exposure had no toxic effect on the fish at the concentrations tested.
The low and high concentration differed in the BCF by an approximate factor of 1.5. The BCF was greater than 10, thus, the uptake of the
substance in fish is established to be significant according to the guideline.

Executive summary:

Regorafenib is a multi-kinase inhibitor for cancer therapy. The aim of this study was to identify the bioaccumulation potential of regorafenib in fish. The study was conducted in accordance with the test guideline OECD 305.

The test consisted of two phases: the exposure (uptake) and post-exposure (depuration) phase.

During the uptake phase, separate groups of fish of the species Lepomis macrochirus were

exposed to the test substance. During depuration, the substance delivery was discontinued and

only tap water was provided.

The fish were exposed to 14C labeled regorafenib at two concentrations (nominally 0.16 and

1.6 ¿g/L) and additionally to the control for a period of 28 days. Subsequently, the fish were

depurated over a period of 14 days. Two replicate tanks for the test concentrations and one tank for the control were used.

A stock solution with a nominal concentration of regorafenib (as ethanolic solution) of 0.8 mg/L for group 2 and 8 mg/L for group 3 were prepared. The stock solutions were delivered continuously to the tanks.

80 fish were used for the test solutions and 10 for the vehicle control group. Mortalities and visible abnormalities of the fish were recorded daily over the uptake and depuration phase.

The hydrographic parameters were recorded weekly. The oxygen concentration should be > 60 % of the air saturation value, the temperature should be maintained at 20 ¿ 25°C and the pH-value between 6.0 and 8.5. These parameters were met.

The concentration of the test substance in the fish and in water was determined through both phases of the test by measurement of the radioactivity. The 14-C concentration in the fish was analyzed by liquid scintillation after liquid degradation of the fish by oxidation (SOLUENE-350) in samples taken on day 4, 6, 10, 14, 21 and 28 (uptake) and on day 30, 32, 38 and 42

(depuration). The 14-C concentration in the water was analyzed by liquid scintillation in samples taken on the

same time points. The bioconcentration factor in fish (BCF) was calculated as the ratio of the mean values of the

14-C concentration in fish and in water, taken at steady state.

In group 2 (low concentration, 0.16 ¿g/L) the mean concentration of regorafenib in water was 0.05 ¿g/L. At steady state, i.e. at sampling days 14 - 28 in group 2, the concentration in fish was 143.72 ¿g/kg. In group 3 (high concentration, 1.6 ¿g/L) the mean value in water was 0.45 ¿g/L. At steady state, i.e. at sampling day 14 - 28 in group 3, the concentration in fish was 1999.84 ¿g/kg.