N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-2,6-difluorobenzamide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2003 - 19 October 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

distribution

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of the study was to determine the levels of the test material in Sprague-Dawley dams’ plasma and milk, in the plasma of foetuses and in the plasma of nursing pups. Milk samples were collected from the dams exposed to 100 mg/kg/day of the test material in diet for 28 weeks as part of a one-generation reproduction toxicity study. Milk and blood samples were collected from the nursing rats on lactation day 9 - 10. Blood samples were also collected from the postnatal day 4 pups and on gestational day 21 foetuses and dams. Both blood and milk samples were analysed for the test material using gradient high performance liquid chromatography - negative ion electrospray ionisation – mass spectrometry.

GLP compliance:

yes

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 6 weeks
- Fasting period before study: No
- Housing: Animals were housed one per cage (pre-breeding) or two per cage (one male and one female during breeding) in stainless steel cages. Dams were housed one per cage (with their litter) in plastic cages provided with corn cob nesting material from approximately day 19 of gestation and throughout the lactation phase of the study. Cages had wire-mesh floors and were suspended above catch pans. Cages contained feed containers and pressure activated, nipple-type watering systems.
- Diet: ad libitum
- Water: municipal water provided ad libitum
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 40 - 70 % (relative)
- Air changes: Approximately 12 - 15 times/hour
- Photoperiod: A 12-hour light/dark photocycle was maintained with lights on at 06:00 and off at 18:00

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Prepared using a dietary pre-mix.

Duration and frequency of treatment / exposure:

Dams were exposed daily for 28 weeks

Dose / conc.:

100 mg/kg diet

No. of animals per sex per dose / concentration:

Not reported

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose administered was the highest dose provided in feed in a one-generation developmental toxicity study.

Details on dosing and sampling:

- Milk samples: Milk samples were collected on gestation day 9 - 10 from dams that had been administered 100 mg/kg/day in a one generation reproductive toxicity study.
- Blood samples: Blood samples were collected on gestation day 9 - 10. They were also obtained from nursing dams on gestational day 21 and postnatal day 4. Blood samples were also collected from gestational day 21 foetuses.
- Sample preparation: Blood samples were centrifuged to obtain plasma. All samples were stored at -80 °C until analysis.
- Sample analysis: Both blood and milk were analysed for the parent compound only; the test material is known to be negligibly metabolised by rats. Milk and plasma samples were removed from the freezer and allowed to thaw. The vials were vortex mixed (~30 seconds) and an aliquot of each sample (100 µL) was transferred to a high-recovery autosampler vial and fortified with radiolabelled test material (¹³C and ¹⁵N, internal standard). 200 µL of acetonitrile was added to each vial, and vortex mixed for ~1 minute. The vials were centrifuged for 10 minutes at 1322 x g for 10 minutes and the liquid was transferred to a clean vial for analysis by gradient high performance liquid chromatography - negative ion electrospray ionisation - mass spectrometry.

ANALYTICAL CONDITIONS
> HPLC-NESI-MS QUANTITATIVE ANALYSIS CONDITIONS
- HPLC System: Hewlett Packard 1100
- Injection Volume: 25 μL
- Guard Column: YMC ODS-AQ, 5 μm, cartridge
- Analytical Column: YMC ODS-AQ, 5 μm, 50 x 3 mm
- HPLC Eluent: A = MilliQ water + 0.1 % acetic acid; B = Acetonitrile + 0.1 % acetic acid
- Gradient: at time 0.00: 90 % A, 10 % B; at time 4.5 and 6.5: 0 % A, 100 % B; at time 7.5 and 10.5: 90 % A, 10 % B. The flow was 0.800 mL/min
- Eluent Split: 15/85 to MS/UV
- UV 1100 VWD: 254 nm
- 1100 MSD Mode: Negative ESI
- Drying Gas: N₂ at 7 L/min and 350 °C
- Nebulizer Pressure: 30 psig
- Capillary Voltage: 4000 V
- Fragmentor: 70 V
- EMV: ~2300 V; EMV Gain: 22
- SIM Ions: 527, 533
- Actual Dwell for all ions: 589 msec

>HPLC-NESI-MS QUALITATIVE (SCAN) ANALYSIS CONDITIONS
Same as above with the following exceptions:
- Guard Column: YMC ODS-AQ, 5 μm, cartridge (20 mm)
- Analytical Column: YMC ODS-AQ, 5 μm, 250 x 4.6 mm
- Fragmentor - 90 V
- EMV: ~2300 V; EMV Gain: 10
- Scan Range: m/z 100 to 800
- Threshold: 20
- Step Size: 0.10

Statistics:

Descriptive statistics were used (mean ± standard deviation).

Key result

Test no.:

#1

Transfer type:

blood/placenta barrier

Observation:

slight transfer

Details on excretion:

The circulating plasma levels of the test material in the dams during pregnancy on gestational day 21 was 18 ± 5 µg/mL, which was 3-fold higher than the levels found in the foetuses (6 ± 1 µg/mL). Concentration in the dams’ plasma dropped by almost 3-fold during nursing (7 ± 1 µg/mL) during lactation days 9-10 after delivering the pups, which was attributed to lactation. The concentration found in milk was 23-fold higher (169 ± 35 µg/mL). Most of the test material was found to transfer to the pups through nursing. The plasma levels found in pups at postnatal day 4 were 8-9 fold higher than the foetuses at gestational day 21. There was no gender difference observed in the plasma levels of the pups. The concentration of the test material in the postnatal day 4 male and female pups was 48 ± 4 and 52 ± 9 µg/mL, respectively.

Metabolites identified:

not measured

Table 1: Results of Analysis of Milk and Plasma Samples

Sample Time	Sample	Matrix Type	Concentration of Test Material µg/mL
Gestation day 21	2968 Dam	Plasma	16.6
	2968 Foetus	Plasma	5.45
	2972 Dam	Plasma	24.7
	2972 Foetus	Plasma	8.16
	2975 Dam	Plasma	13.3
	2975 Foetus	Plasma	6.06
	2979 Dam	Plasma	16.0
	2979 Foetus	Plasma	5.15
Postnatal day 4	2978 male pup	Plasma	43.7
	2978 female pup	Plasma	42.5
	2984 male pup	Plasma	52.1
	2984 female pup	Plasma	60.0
	2986 male pup	Plasma	45.7
	2986 female pup	Plasma	59.1
	2993 male pup	Plasma	50.0
	2993 female pup	Plasma	45.0
Lactation day 9-10	2983 Dam	Plasma	7.33
		Milk	216
	2984 Dam	Plasma	8.38
		Milk	176
	2986 Dam	Plasma	7.11
		Milk	137
	2992 Dam	Plasma	6.34
		Milk	148

 

Table 2: Summary of results

Sample	Concentration of Test Material (µg/mL) in Sample
	Mean	SD
Dam Plasma Gestation Day 21	17.65	4.91
Foetus Plasma Gestation Day 21	6.21	1.36
Pup Plasma Postnatal Day 4 - Males	47.88	3.85
Pup Plasma Postnatal Day 4 - Females	51.65	9.19
Dam Plasma Lactation Day 9-10	7.29	0.84
Dam Milk Lactation Day 9-10	169.25	35.23

 

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
The data indicate that the exposure of the test material to foetuses through the placenta is limited and the majority of exposure occurs postnatally through nursing. The systemic exposure of neonates through milk surpassed the systemic exposure to non-lactating dams by approximately 3-fold.

Executive summary:

The purpose of the study was to determine the levels of the test material in Sprague-Dawley dams’ plasma and milk, in the plasma of foetuses and in the plasma of nursing pups. Milk samples were collected from the dams exposed to 100 mg/kg/day of the test material in diet for 28 weeks as part of a one-generation reproduction toxicity study. Milk and blood samples were collected from the nursing rats on lactation day 9-10. Blood samples were also collected from the postnatal day 4 pups and on gestational day 21 foetuses and dams. Both blood and milk samples were analysed for the test material using gradient high performance liquid chromatography - negative ion electrospray ionisation – mass spectrometry.

The circulating plasma levels of the test material in the dams during pregnancy on gestational day 21 was 18 ± 5 µg/mL, which was 3-fold higher than the levels found in the foetuses (6 ± 1 µg/mL). Concentration in the dams’ plasma dropped by almost 3-fold during nursing (7 ± 1 µg/mL) during lactation days 9-10 after delivering the pups, which was attributed to lactation. The concentration found in milk was 23-fold higher (169 ± 35 µg/mL). Most of the test material was found to transfer to the pups through nursing. The plasma levels found in pups at postnatal day 4 was 8-9 fold higher than the foetuses at gestational day 21. There was no gender difference observed in the plasma levels of the pups. The concentration of the test material in the postnatal day 4 male and female pups was 48 ± 4 and 52 ± 9 µg/mL, respectively.

The data indicate that the exposure of the test material to foetuses through the placenta is limited and the majority of exposure occurs postnatally through nursing. The systemic exposure of neonates through milk surpassed the systemic exposure to non-lactating dams by approximately 3-fold.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 2001 - 13 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

absorption

excretion

Qualifier:

no guideline followed

Principles of method if other than guideline:

A limited pharmacokinetic study was conducted in Fischer 344 rats to provide data on the absorption of the radiolabelled test material to assist in dose level selection for a two-year rat chronic toxicity study.
Two male and two female Fischer 344 rats per dose were administered a single oral dose of 10, 100 or 250 mg/kg of radiolabelled test material. Urine was collected at 8, 24, 48 and 72 hours post dosing, and faeces were collected at 24, 48 and 72 hours and analysed for radioactivity. Blood was collected at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 24, 32, 48 and 72 hours post dosing. Radioactivity in the plasma was determined and concentration-time course profiles were constructed.

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

¹⁴C

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: Males 194 - 204 g; Females 186 - 192 g
- Fasting period before study: Animals were restricted to two pellets of feed for 16 hours prior to dosing. Animals had free access to food immediately following dosing.
- Housing: All-glass Roth type metabolism cages
- Individual metabolism cages: yes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least one day prior to use
-Cannulation: Animals were provided cannulated in the jugular vein by the supplier. The cannulae were surgically exteriorised on the day of receipt.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 % RH
- Air changes: Air was drawn through the metabolism cages at approximately 500 mL/min
- Photoperiod (hrs dark / hrs light): 12 hour photocycle

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % METHOCEL cellulose ethers

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The doses were prepared as aqueous suspensions in 0.5 % METHOCEL cellulose ethers. A non-radiolabelled stock solution of the test material at 100 milligram per gram of METHOCEL was prepared in 0.5 % METHOCEL. A suspension was achieved by stirring with a magnetic stirrer for 24 hours at 4 °C. Serial dilutions were then prepared. Appropriate amounts of radiolabelled test material were added to each dose to obtain a target radioactivity of 50 µCi/g METHOCEL. The dose solutions were continuously mixed for homogeneity with a magnetic stirrer until dosing.

VEHICLE
- Concentration in vehicle: 2, 20 and 50 mg test material per g of METHOCEL
- Amount of vehicle (if gavage): 5 mL/kg bw

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
Homogeneity of the dose was determined by liquid scintillation spectrometry. Aliquots of the dose were taken from different locations in the container and analysed. The concentration of the test material in the dosing solutions was determined by HPLC analysis with UV detection. Dose solutions were diluted with acetonitrile prior to HPLC analysis. The radioactivity of the dose was confirmed by liquid scintillation analysis.

Duration and frequency of treatment / exposure:

A single dose was administered.

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

2 animals per sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose levels for this study were based on previous toxicity information. The low dose in this study was selected on the basis that it was 10-fold lower than the NOEL reported in a 4 week dietary toxicity study. The high dose selected was half of the level where effects (increased liver and kidney weights) were observed in the same study. These doses were considerably below the reported oral LD50 of this substance in Fischer 344 rats.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma and cage washes
- Time and frequency of sampling: Urine was collected at 8, 24, 48 and 72 hours post dosing and the cages were rinsed with water and the cage wash collected. Faeces were collected at 24, 48 and 72 hours. Blood (0.1 mL) was collected at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 24, 32, 48 and 72 hours post dosing from the jugular cannula.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: Samples collected 8 - 24 hours post dosing
- From how many animals: Equal volume aliquots of urine samples from each collection interval were pooled by dose and by sex for analysis. Those with the highest amount of radioactivity were used for metabolite identification
- Method type(s) for identification: HPLC/LCQ-MS/RAM. Reversed phase high performance liquid chromatography with column effluent split into two streams. The first, directed to a radioactivity monitor (RAM) and the second to a mass spectrometer equipped with a Finnigan MAT electrospray interface operated in negative electrospray ionisation mode (NESI).
- Limits of detection and quantification: Samples with dpm less than twice the concurrently run background (blanks) were considered to contain insufficient radioactivity to reliably quantify. For non-quantifiable excreta samples a value of '0' was used in calculations. Non-quantifiable plasma samples were assigned the quantitation limit (QL) for calculations. QL (as a fraction of administered dose) were calculated using the following formula:
QL = (Background dpm/aliquot weight) x (dilution factor/dose dpm)

Statistics:

Pharmacokinetic analyses were performed on the plasma concentration-time data to calculate the area under the concentration-time curves (AUC) and half-life of elimination of radioactivity derived from radiolabelled test material using WinNonlin® pharmacokinetic modelling program (Pharsight Corp., Mountain View, California).

Type:

absorption

Results:

The test material was readily absorbed through the GI tract

Type:

absorption

Results:

5 to 30-fold radioactivity above the background was recorded within 5 minutes of dosing

Type:

absorption

Results:

Peak plasma concentrations were reached between 3 and 6 hours

Type:

excretion

Results:

The t½,β elimination of the test material radioactivity from plasma was found to be 52, 63 and 54 hours in males and 61, 61 and 57 hours in females dosed at 10, 100 and 250 mg/kg, respectively.

Type:

excretion

Results:

Urinary excretion was negligible at all levels. The primary route of excretion was faecal.

Details on absorption:

The test material was rapidly absorbed from the GI tract in both sexes at all three doses. Within 5 min after gavage, 5 to 30-fold higher than background radioactivity was found in plasma. The peak plasma concentrations (Cmax) were reached within 3 to 6 hours post dosing. There was no dose or gender related difference in the time taken to achieve peak plasma concentrations (Tmax).
There were no dose proportionalities in the AUC and Cmax among doses. A 9.1 to 9.2-fold increase in dose from the target dose of 10 to 100 mg/kg produced AUCs that were only 6 to 6.6-fold larger. Similarly, when the dose was increased by 25 to 28-fold (from the target dose of 10 to 250 mg/kg) AUCs were increased only by 7 to 9- fold. Similar patterns were observed when Cmax among the doses were compared: a 6 to 7.5-fold increase in Cmax between the target doses of 10 and 100 mg/kg, and an 8 to 9-fold increase in Cmax between the target doses of 10 and 250 mg/kg. The increase in dose was 173 to 199 % between the target doses of 100 and 250 mg/kg; however increase in the AUC was only between 20 and 36 %. Similarly, increase in the Cmax between the two doses (100 and 250 mg/kg) was only 25 % for both sexes. A less than proportional increase in the AUCs and Cmax versus the increase in dose suggests saturation of absorption from the GI tract at higher doses. The t½,β of elimination of the test material-derived radioactivity from plasma was calculated from 2 rats per dose group and found to be 52, 63, and 54 hours for males and 61, 61, and 57 hours for females at the target doses of 10, 100, and 250 mg/kg, respectively.

Details on excretion:

Urinary excretion was negligible at all dose levels administered, both in male and female rats. Only a fraction (an average of 0.7 - 5.0 %) of the administered radioactivity was recovered in the urine by the end of the study. Conversion of the total amount (as percent of administered dose) of administered radioactivity recovered in urine by 72 hours demonstrated that the 100 and 250 mg/kg target doses were identical (0.33 vs. 0.34 mg-equivalent for males and 0.43 vs. 0.42 mg-equivalent for females). Renal clearance of radioactivity dropped at the highest dose when compared to both middle and lowest doses. An identical amount of urinary excretion of test material-derived radioactivity at two doses that were 2.7 to 3.0-fold apart (between the target doses of 100 and 250 mg/kg) and a drop in urinary clearance are also indications of saturation of absorption and/or metabolism in animals.
The primary route of excretion was faecal. Males eliminated 53, 61, and 69 % and females eliminated 58, 90, and 80 % of dose in the faeces at the target doses of 10, 100, and 250 mg/kg, respectively. In males, faecal elimination of radioactivity occurred rapidly with 88 - 95 % of the total excreted radioactivity recovered within 24 hours. In females, 54 - 77 % of the eliminated radioactivity was recovered within 24 hours post-dosing and 96 % within 48 hours.

Test no.:

#1

Toxicokinetic parameters:

AUC: 8.28 µg h/mL (male rats at 10 mg/kg bw)

Test no.:

#2

Toxicokinetic parameters:

AUC: 54.68 µg h/mL (male rats at 100 mg/kg bw)

Test no.:

#3

Toxicokinetic parameters:

AUC: 74.32 µg h/mL (male rats at 250 mg/kg bw)

Test no.:

#4

Toxicokinetic parameters:

AUC: 13.15 µg h/mL (female rats at 10 mg/kg bw)

Test no.:

#5

Toxicokinetic parameters:

AUC: 80.34 µg h/mL (female rats at 100 mg/kg bw)

Key result

Test no.:

#6

Toxicokinetic parameters:

AUC: 96.55 µg h/mL (female rats at 250 mg/kg bw)

Test no.:

#1

Toxicokinetic parameters:

Cmax: 0.34 µg/mL (male rats at 10 mg/kg bw)

Test no.:

#2

Toxicokinetic parameters:

Cmax: 2.16 µg/mL (male rats at 100 mg/kg bw)

Test no.:

#3

Toxicokinetic parameters:

Cmax: 2.70 µg/mL (male rats at 250 mg/kg bw)

Test no.:

#4

Toxicokinetic parameters:

Cmax: 0.43 µg/mL (female rats at 10 mg/kg bw)

Test no.:

#5

Toxicokinetic parameters:

Cmax: 3.20 µg/mL (female rats at 100 mg/kg bw)

Key result

Test no.:

#6

Toxicokinetic parameters:

Cmax: 4.00 µg/mL (female rats at 250 mg/kg bw)

Test no.:

#1

Toxicokinetic parameters:

Tmax: 3.00 hrs (male rats at 10 mg/kg bw)

Test no.:

#2

Toxicokinetic parameters:

Tmax: 6.00 hrs (male rats at 100 mg/kg bw)

Test no.:

#3

Toxicokinetic parameters:

Tmax: 4.50 hrs (male rats at 250 mg/kg bw)

Test no.:

#4

Toxicokinetic parameters:

Tmax: 5.50 hrs (female rats at 10 mg/kg bw)

Key result

Test no.:

#5

Toxicokinetic parameters:

Tmax: 6.00 hrs (female rats at 100 mg/kg bw)

Test no.:

#6

Toxicokinetic parameters:

Tmax: 4.50 hrs (female rats at 250 mg/kg bw)

Metabolites identified:

yes

Details on metabolites:

The following metabolites were tentatively identified in urine:
1. Glucuronide conjugate of hexafluoroalkoxy-fluorodichloroaniline (co-eluted with metabolite two, retention time 6.2 minutes)
2. Mercapturic acid conjugate of the parent compound (co-eluted with metabolite 1, retention time 6.0 minutes)
3. Sulfate conjugate of hexafluoroalkoxy-fluorodichloroaniline (retention time 10.8 minutes).
No parent compound was detected in the urine samples. Metabolites 1 and 2 accounted for 35 % of the excreted radioactivity with metabolite 3 representing 65 % of the excreted radioactivity.

Bioaccessibility (or Bioavailability) testing results:

The non-proportionalities in AUC and Cmax and higher faecal elimination, especially at higher doses, indicate a saturation of absorption of the test material from the GI tract. Saturation of absorption from the GI tract decreased bioavailability at the target doses of 100 and 250 mg/kg. The administered dose at 250 mg/kg was 173–199 % higher than the target dose of 100 mg/kg dose. However, this increase in dose resulted in an increase of only 20–36 % in AUC and 25 % in Cmax. A decrease in absorption/ bioavailability was also apparent from a limited (almost identical) amount of urinary excretion at the higher doses which were approximately 3-fold apart (actual difference between 100 and 250 mg/kg).

Table 2: Concentration Time-Course of Test Material Derived Radioactivity in Plasma

Group (mg/kg)	Sex	Animal No.	Time (hours)
			0.08	0.25	0.5	1	2	3	4	6	8	24	32	48	72
10	M	1	NQ (0.011)	0.016	0.032	0.126	0.256	0.310	0.301	0.228	0.185	0.096	0.077	0.032	0.050
		2	NQ (0.007)	0.007	0.030	0.172	0.272	0.368	0.360	0.321	0.250	0.130	0.129	0.082	0.077
		Mean	NQ (<0.009)	0.012	0.031	0.149	0.264	0.339	0.331	0.275	0.217	0.113	0.103	0.057	0.063
	F	1	NQ (<0.009)	0.013	0.031	0.097	0.349	0.389	0.363	0.392	0.363	0.119	0.114	0.088	0.058
		2	NQ <0.012)	0.004	0.012	0.071	0.379	0.433	0.404	0.452	0.461	0.229	0.155	0.154	0.129
		Mean	NQ (<0.010)	0.009	0.021	0.084	0.364	0.411	0.383	0.422	0.412	0.174	0.134	0.121	0.094
100	M	1	NQ (0.234)	NQ (0.170)	0.166	0.768	1.691	2.375	1.797	2.389	1.691	0.640	0.558	0.417	0.432
		2	NQ (0.158)	NQ (0.075)	0.438	0.840	1.534	1.765	1.576	1.926	1.574	0.826	0.591	0.398	0.371
		Mean	NQ (<0.196)	NQ (<0.123)	0.302	0.804	1.612	2.070	1.686	2.157	1.633	0.733	0.574	0.407	0.402
	F	1	NQ (0.160)	NS	0.331	1.230	2.016	1.921	1.830	2.119	1.677	0.762	0.458	0.499	0.407
		2	NQ (0.154)	0.209	0.477	0.969	2.641	3.190	3.620	4.276	3.487	1.444	1.102	0.698	0.608
		Mean	NQ (<0.157)	0.209	0.404	1.100	2.329	2.555	2.725	3.197	2.582	1.103	0.780	0.599	0.507
250	M	1	0.11	NQ (0.275)	0.475	0.956	1.851	2.657	2.938	3.820	2.279	1.164	1.016	0.771	0.743
		2	NQ (0.064)	0.167	0.487	0.762	1.262	1.572	1.371	1.500	1.234	NS	NS	NS	0.340
		Mean	NQ (<0.088)	NQ (<0.221)	0.481	0.859	1.556	2.114	2.154	2.660	1.757	1.164	1.016	0.771	0.542
	F	1	NQ (0.103)	0.282	0.488	0.957	2.068	3.115	2.981	2.694	1.880	1.151	0.759	0.778	0.643
		2	NQ (0.273)	0.375	0.850	0.973	3.882	4.483	4.389	4.883	3.321	1.621	1.161	0.830	0.690
		Mean	NQ (<0.188)	0.328	0.669	0.965	2.975	3.799	3.685	3.789	2.601	1.386	0.960	0.804	0.666

NQ = Non-quantifiable. Non-quantifiable plasma samples were assigned the quantitation limit (QL) for calculations and displayed as NQ with the quantitation limits in parentheses

NS = No sample

 

Table 3: Comparison of Dose, AUC, Plasma Cmax, Plasma Clearance and Plasma Half-Life

	Male			Female
Target Dose (mg/kg)	10	100	250	10	100	250
Actual dose (mg/kg)	9.52	86.49	236.12	8.47	78.10	233.54
Actual Dose ratio*	-	9.09	24.80	-	9.22	27.57
AUC (µg h/mL)	8.28	54.68	74.32	13.15	80.34	96.55
AUC Ratio**	-	6.61	8.98	-	6.11	7.34
Cmax (µg/mL)	0.34	2.16	2.70	0.43	3.20	4.00
Cmax Ratio ***	-	6.35	7.93	-	7.53	9.40
Tmax (h)	3.00	6.00	4.50	5.50	6.00	4.50
t1/2,β	51.60	63.24	53.98	60.97	60.73	56.50
% Dose in Urine	2.11	1.94	0.72	4.56	3.04	0.98
Total mg-eq. in Urine	0.04	0.33	0.34	0.07	0.43	0.42
Clr (mL/h)	4.92	6.11	4.55	5.53	5.66	5.00

*Dose ratio = actual dose/low dose (10 mg/kg)

**AUC = actual AUC/low dose AUC

***Cmax ratio = actual Cmax/low dose Cmax

 

Table 4: Recovery of Test Material Derived Radioactivity in Urine

Group (mg/kg)	Sex	Animal No.	0 - 8 hrs	0 - 24 hrs	0 - 48 hrs	0 - 72 hrs	Total mg eq. recovered
							0 - 72 hrs
10	Males	1	0.25	1.03	1.48	1.72	0.03
		2	0.05	1.38	2.11	2.51	0.05
		Mean	0.15	1.20	1.79	2.11	0.04
	Females	1	0.33	1.93	2.42	2.93	0.05
		2	NQ	2.91	4.64	5.85	0.08
		Mean	0.33	2.58	3.69	4.56	0.07
100	Males	1	0.01	1.22	1.62	1.84	0.31
		2	0.13	1.44	1.79	2.03	0.36
		Mean	0.07	1.33	1.71	1.94	0.33
	Females	1	0.00	1.17	1.56	1.75	0.30
		2	0.43	2.97	3.48	4.33	0.55
		Mean	0.22	2.07	2.52	3.04	0.43
250	Males	1	0.13	0.51	0.74	0.89	0.44
		2	0.05	0.33	0.47	0.55	0.24
		Mean	0.09	0.42	0.60	0.72	0.34
	Females	1	0.00	0.46	0.70	0.84	0.41
		2	0.06	0.54	0.89	1.06	0.42
		Mean	0.06	0.53	0.82	0.98	0.42

NQ =Non-quantifiable

Table 5: Recovery of Test Material Derived Radioactivity in Faeces

Group (mg/kg)	Sex	Animal No.	0 - 24 hrs	0 - 48 hrs	0 - 72 hrs	Total mg eq. recovered
						0 - 72 hrs
10	Males	1	52.75	56.67	57.98	1.13
		2	40.73	46.88	48.80	0.94
		Mean	46.74	51.78	53.39	1.03
	Females	1	47.81	55.88	57.83	1.04
		2	15.65	32.65	57.25	0.80
		Mean	31.73	44.27	57.54	0.92
100	Males	1	50.45	52.69	53.54	8.93
		2	65.91	68.35	69.44	12.39
		Mean	58.18	60.52	61.49	10.66
	Females	1	54.86	68.94	69.98	12.02
		2	84.39	99.70	109.83	14.04
		Mean	69.63	84.32	89.90	13.03
250	Males	1	65.34	71.74	72.37	35.89
		2	56.87	64.66	65.28	29.15
		Mean	61.11	68.20	68.82	32.52
	Females	1	28.68	61.03	62.21	30.57
		2	57.24	92.78	97.94	39.04
		Mean	42.96	76.91	80.07	34.80

Conclusions:

Interpretation of results: bioaccumulation potential cannot be judged based on study results
There were no dose proportionalities in the area under the concentration-time course profiles (AUC) or the maximum plasma concentrations (Cmax) among doses. The primary route of excretion was determined to be through faecal elimination.

Executive summary:

A limited pharmacokinetic study was conducted in Fischer 344 rats to provide data on the absorption and elimination of the radiolabelled test material to assist in dose level selection for a two-year rat chronic toxicity study. The study was carried out under GLP conditions.

Two male and two female Fischer 344 rats per dose were administered a single oral dose of 10, 100 or 250 mg/kg of radiolabelled test material. Urine was collected at 8, 24, 48 and 72 hours post dosing and faeces were collected at 24, 48 and 72 hours and analysed for radioactivity. Blood was collected at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 24, 32, 48 and 72 hours post dosing. Radioactivity in the plasma was determined and concentration-time course profiles were constructed.

There were no dose proportionalities in the area under the concentration-time course profiles (AUC) or the maximum plasma concentrations (Cmax) among doses. The primary route of excretion was determined to be through faecal elimination. Within 72 hours, 53 - 90 % of the administered dose was recovered in the faeces. Males eliminated 53 - 69 % and females eliminated 58 - 90 % of dose in the faeces. Faecal elimination was rapid in males with 88-95 % of the eliminated radioactivity recovered within 24 hours. In females, 54 -77 % of the total faecal elimination of radioactivity was recovered within 24 hours and 77 - 96 % within 48 hours. At all dose levels, only 0.7 - 5.0 % was recovered in the urine. The total urinary excretion through urine by 72 hours post dosing was identical at the highest two dose levels. The similar levels of recovery and the drop in urinary clearance observed at the highest two dose levels were indications of saturation of absorption and/or metabolism in animals. Similarly, non-proportionalities in AUC and Cmax and higher faecal elimination suggest saturation of absorption from the GI tract that caused a decrease in bioavailability at 100 and 250 mg/kg. Three urinary metabolites were tentatively identified using HPLC/LC-MS analysis; these metabolites include the glucuronide and sulfate conjugates of hexafluoroalkoxy-fluorodichloroaniline, as well as a mercapturic acid conjugate of the parent compound. No differences were observed in the profile of urinary metabolites between sexes or dose levels.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2002 - 16 December 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

other: pharmacokinetics and metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

¹⁴C

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 - 12 weeks old
- Weight at study initiation: males 166 - 230 g; females 117 - 152 g
- Fasting period before study: 16 hours prior to dosing and 4 hours post dosing
- Housing: During acclimation animals were housed two per cage in stainless steel cages and for at least 2 days in individual metabolism cages. The stainless steel cages had wire- mesh floors suspended above catch pans. Cages contained a hanging feeder and a pressure activated nipple type watering system.
Following administration of the test material, animals were housed one per cage in glass Roth-type metabolism cages. The metabolism cages were designed for the separation and collection of urine and faeces as well as expired air. Air was drawn through the metabolism cages at ~500 mL/minute.
- Individual metabolism cages: yes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least one week, with at least two days acclimating to metabolism cages. The test material had previously been demonstrated to be eliminated predominantly via the faeces; when high levels of radioactivity were observed in the skin of animals dosed with 100 mg/kg, it was considered to be due to contamination. The test group was therefore repeated with all rats fitted with dermal jackets to avoid contamination of the fur. Animals were acclimated to these jackets during the last four days of the acclimation period.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 12 - 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hour photocycle

Route of administration:

other: oral gavage and intravenously

Vehicle:

other: oral gavage: 0.5 % aqueous METHOCEL cellulose ether; intravenous: Prepared in a dosing emulsion (please refer to details on exposure below)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- Oral:
For oral dosing, suspensions of ¹⁴C-radiolabelled test material were prepared in the following manner: No unlabelled material was required to obtain the target concentration for the low dose, while for the high dose solution, a measured amount of unlabelled test material was placed in a vial. A suitable amount of radiotracer, dissolved in acetone, was added to the flasks, and the acetone removed by drying under a gentle stream of nitrogen. Crystals of test material were dissolved in ethanol (≤ 1 and 6.2 4% w/w of the dose suspension for the low and high doses, respectively). The required amount of an aqueous solution of 0.5 % METHOCEL was then added and the suspensions of ¹⁴C-test material were stirred at 4 °C for several (2 - 7) days prior to the administration to the animals. The non-radiolabelled suspension used for repeated dosing was prepared in two batches (7 days apart) by adding a measured amount of unlabelled test material to a vial, followed by adding ethanol (≤ 1 % w/w of the final volume of the dose suspension) to dissolve the test material and the required amount of 0.5 % METHOCEL. The suspension of unlabelled test material was continuously stirred at 4 °C for the duration of the repeated dose administration period. The first batch of the dose suspension was used for days 1 - 7 of the dosing period while the second batch was used for days 8 - 14 of the dosing period.
Animals were dosed at a target volume of 5 mL dose suspension per kg of body weight.

- Intravenous:
For IV injection, the radiolabelled dose emulsion was prepared as described by Weber et al. (1993). Briefly, the required amount (3.4 mg) of ¹⁴C-test material dissolved in acetone was added to a tall glass vial and the acetone removed by drying under a gentle stream of nitrogen. Twenty-six milligrams of L-α-phosphatidylcholine, prepared in chloroform/methanol; 9: 1, was added to the vial and solvent evaporated with gentle stream of nitrogen gas. Corn oil (0.75 g) was added to the vial and test material and L-α-phosphatidylcholine was dissolved by stirring and slight heating. In a separate vial 26 mg of cholic acid was dissolved in 7.3 mL of Ringer's solution with the use of 1 N NaOH (ca. 100 µL) and stirring/slight heating. Ringer's solution containing cholic acid and NaOH was added to the vial containing test material, L-α-phosphatidylcholine and corn oil and the vial stirred at low heat for 45 minutes. The mixture was emulsified with a polytron® homogeniser for two minutes at maximum speed and finally by sonicating for 3 minutes using a Branson Sonfier at the output control setting of 5. The pH of the emulsion was determined to be approximately 8. The emulsion was kept refrigerated and brought to room temperature before injection. This emulsion has been reported to be stable for at least 3 months when kept refrigerated (Weber et al., 1993). The dose emulsion was used within 24 hours after preparation after determining concentration of radioactivity and homogeneity right (ca.1 hour) before dosing.
Animals were dosed at a target volume of 2.5 mL dose emulsion per kg of body weight.

DOSE CONFIRMATION
The concentration of test material in the dose suspension/emulsion was determined by HPLC with a UV detector. Aliquots of low and high dose suspensions and low dose emulsion were diluted with acetonitrile prior to HPLC analysis. The concentration was quantified using unlabelled standards.
Radioactivity of the dose solutions was determined by liquid scintillation counting (LSC) of aliquots of the dosing solutions.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
LSC analyses of aliquots of the dose solution taken from various locations in the solution containers were used to confirm homogeneity of the dosing solutions containing ¹⁴C-test material. For the non-radiolabelled dose suspensions, homogeneity was established by determining concentration in two aliquots taken from different locations in the solution containers.
The stability of test material in 0.5 % aqueous METHOCEL was determined in the unlabelled repeated dose solution on day 7 (this study) and day 12 (earlier oral gavage study) after the preparation of the dose suspension. Oral dose suspensions were used within 2 - 7 days of preparation. The IV dose emulsion was used within 24 hours of preparation.

Duration and frequency of treatment / exposure:

- Groups 1, 2 and 3 were administered a single oral dose. These animals were sacrificed 7 days after the dose.
- Group 4 was administered with 14 daily oral doses of unlabelled test material via gavage and a dose of labelled test material on day 15. These animals were sacrificed 7 days after the final dose.
- Group 5 was administered with a single intravenous dose. These animals were sacrificed 7 days after the dose.

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

Group 5: fluorodichlorophenyl ring labelled test material

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

Groupe 4: 14 daily doses of unlabelled test material at 1 mg/kg bw, and 1 mg/kg bw of fluorodichlorophenyl ring labelled test material on day 15

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

Group 3: difluorobenzoyl ring labelled test material

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2: fluorodichlorophenyl ring labelled test material

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

Group 1: fluorodichlorophenyl ring labelled test material

No. of animals per sex per dose / concentration:

- Groups 1, 2 and 4 consisted of 4 rats per sex per dose.
- Group 3 consisted of 4 male rats per dose.
- Group 5 consisted of 3 rats per sex per dose.

Control animals:

no

Details on dosing and sampling:

DOSE ADMINISTRATION
- Oral:
The rats were weighed prior to dosing with ¹⁴C-test material and on days 1 and 8 of the multiple dosing period (Group 4). For oral dosing, based on their body weight, a measured volume of dose solution was administered by gavage.
- Intravenous:
For IV injection (Group 5), rats were placed under a heat lamp for ca. 1 min while held in a plastic tub containing corn-cob bedding. They were restrained in a polyethylene restrainer (decapicone bag) and placed on their side on a heating pad (37 °C). Based on their body weight, a weighed amount of the dose emulsion was drawn in a 1 mL glass syringe and slowly injected into the lateral tail vein using a 25 G needle. Before injecting the emulsion, proper entry into the vein was verified by checking the flow of blood into the syringe by drawing the plunger back.

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes, volatile organics (group 2) and expired CO₂ (group 2).
The following were sampled from animals of groups 1, 2 and 4: adrenal, brain, fat, GI tract (including ingesta), kidney, liver, ovaries, residual carcass, skin, spleen, testes and uterus.
The following were sampled from group 3: skin and residual carcass.
The following were sampled from group 5: brain, fat, injection site, kidney, liver, skin, spleen and residual carcass.
- Time and frequency of sampling: All urine voided during the study was collected in dry-ice cooled traps at 24 hour intervals. The cages were rinsed with water at the time the traps were changed and the rinse was collected. In some groups, cages were also rinsed with methanol after water rinse at 24 hours to remove glass-bound compound(s), if any, in order to increase the total recovery. However, it was abandoned after the Group 2 experiment, as almost no radioactivity was recovered in the organic rinse. Faeces were collected in dry-ice chilled containers at 24-hour intervals. To measure the volatile organics in animals of group 2, air was drawn through the cage at approximately 500 mL/minute. Upon exiting the cage, the air was passed through charcoal to trap organic volatiles. The charcoal traps were changed at 24 hours. The remaining tissues were collected at necropsy following sacrifice.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces
- From how many animals: Pooled urine and faecal samples (0-24 and 48-72 hr) from 4 male and 4 female Fischer 344 rats orally dosed with 1 or 100 mg 14C-test material/kg bw (Groups 1-4) were submitted for reverse-phase high-performance liquid chromatography (HPLC) analysis for metabolite profiles.
Pooled urine and faecal samples (0-24 and 48-72 hr) from 3 male and 3 female Fischer 344 rats intravenously dosed with 1.0 mg 14C-test material/kg bw (Group 5) were also submitted for HPLC analysis. Equal volumes of the individual urine and faeces were used for pooling the male and female samples. All samples were stored at -80°C until analysed.

Statistics:

Descriptive statistics were used (mean ± standard deviation).
The time-course of radioactivity excreted in urine of all groups and faeces of the IV injected rats (Group 5), which represented biliary excretion of the injected radioactivity, was used to calculate urinary and biliary elimination half-lives. The rate of excretion of radioactivity in urine (Group 1-5) and faeces (Group 5) from individual rats was analysed by a non-compartmental modelling method using a nonlinear least square regression program (WinNonlin, Pharsight Corp., Mountain View, CA) to determine respective elimination half-lives, as described by Gibaldi and Perrier (1982).

Details on absorption:

- Oral Administration
A total of 41 - 55 % of the single oral dose of 1 mg fluorodichlorophenyl ring-label test material/kg (Group 1) was absorbed from the GI tract. Absorption of test material after multiple 1 mg/kg oral doses (Group 4) was lower (35 % of dose) than the single oral dose.
Absorption of the single oral dose of 1 mg/kg ¹⁴C-test material labelled at the difluorobenzoyl ring of the test compound (Group 3) was higher than the fluorodichlorophenyl ring-label test material (Group 1) with ca. 56 % of the dose absorbed.
Absorption of the single oral high dose of 100 mg/kg (Group 2) was only 2 - 5 %.

Details on distribution in tissues:

At 168 hours post-dosing, 17 - 34 % of the low dose test material was recovered with the tissues while only ~1 % of the high dose was recovered with the tissues. There were no gender-related differences in the distribution pattern of radioactivity in different tissues of rats. The level of radioactivity in tissues between low and high doses was not proportional to the difference between doses and mostly accounted for only 4 to 5-fold increase at the high dose. Multiple dosing reduced levels of the final radiolabelled dose in most tissues by an average of 29 - 50 % when compared with the levels after single oral dose.

Details on excretion:

The majority of the test material was eliminated in faeces with no gender-related differences. The mean faecal recovery of the low dose of test material was 29 - 35 % dose within 24-hours post-dosing, somewhat higher than the 24-hour biliary elimination of 13 - 17 % following IV injection. Faecal elimination of 100 mg/kg was 87 - 93 % of the administered dose during the first 24 hours. The rate of biliary elimination of the IV-dosed test material was linear with an elimination half-life of 53 and 60 hours for male and female rats, respectively.
Urinary excretion of the low dose of test material ranged from 10 - 16 % with no gender-related differences regardless of the number of doses or routes of exposure. Urinary elimination of test material labelled on the other ring was 2.3-fold higher. Less than 1.5 % of the high dose of test material was recovered in urine. Urinary elimination was biphasic with an initial fast elimination half-life between 20 and 35 hours, whereas the slow elimination half-life was between 62 and 110 hours.

Metabolites identified:

yes

Details on metabolites:

A total of five metabolites were found in excreta at levels of >5 % of the administered dose. Most of the radioactivity recovered in faeces was parent test material (51 - 61 % and 84 - 100 % of the recovered radioactivity after IV and oral doses, respectively). Four minor metabolic peaks were found in faeces, two of them eluted before and two after the parent compound based on reverse-phase HPLC separation. A total of 2 metabolites were found in urine of rats dosed orally, and a third metabolite was detected in females dosed IV. Three different peaks, all eluting earlier on the reverse-phase HPLC column, were found in urine of animals dosed with test material labelled on the other ring. The test material is either excreted unchanged (primarily via faeces) or metabolised via cleavage of the acyl urea moiety followed by conjugation with glycine and excretion via urine. The corresponding aniline metabolite is hydroxylated and conjugated with sulfate or glucuronic acid.

Dose Solution

For the low dose suspension of 0.20 mg/g METHOCEL, the actual concentration of test material ranged between 0.20 to 0.25 mg test material/g. The concentration of the IV dose emulsion was 0.43 mg test material/g. The high dose suspension was 21.33 mg test material/g. The concentration of radioactivity in each of the oral dose suspensions ranged from 11.1 to 16.1 µCi/g dose suspension. For the IV dose emulsion, the concentration of radioactivity was 32.9 µCi/g. The mean body weight (bw), mean amounts of test material and mean radioactivity administered to each group of rats are presented in Table 1.

Table 1: Mean Body Weight, Amounts of Dose Solution, Radioactivity and Test Material Administered

Group	Sex	Bodyweight (g)	Dose solution (g)	Radioactivity (µCi)	Test material (mg)	Test Material (mg/kg)	Test Material (µCi/kg)
1	M	0.1857	1.1124	12.301	0.278	1.50	66.25
	F	0.1416	0.8324	10.460	0.192	1.36	73.78
2	M	0.1950	0.9566	15.396	20.376	104.61	79.05
	F	0.1210	0.6245	10.051	13.301	109.96	83.09
3	M	0.1717	0.8658	11.721	0.171	1.00	68.23
4	M-unlabelled	0.2110	0.918	-	0.20	0.85 - 0.95	-
	M-labelled	0.2260	1.2002	17.119	0.250	1.10	75.76
	F-unlabelled	0.1426	0.918	-	0.20	0.93 - 1.03	-
	F-labelled	0.1450	0.7912	11.285	0.165	1.13	77.73
5	M	0.1978	0.4665	15.344	0.200	1.01	77.59
	F	0.1391	0.3364	11.066	0.144	1.04	79.55

Table 2: Disposition of Radioactivity Following a Single Oral Dose of 1 or 100 mg ¹⁴C-test material/kg bw 7 days post-dosing (% of administered dose - mean values)

Group	1		2		3	4		5
Sex	Male	Female	Male	Female	Male	Male	Female	Male	Female
Urine & rinse	15.87	11.21	1.53	0.87	37.15	11.30	9.98	14.25	11.84
Faeces	64.43	60.05	89.95	96.11	36.19	51.06	56.21	45.79	41.34
¹⁴CO₂	ND	ND	0.00	0.00	ND	ND	ND	ND	ND
Expired organics	ND	ND	0.00	0.00	ND	ND	ND	ND	ND
Tissues & carcass*	28.52	23.90	1.39	1.16	17.12	17.88	18.79	31.52	33.91
Final cage wash	0.68	0.71	0.57	0.09	1.17	1.96	0.41	0.36	0.44
Others**	4.12	4.53	0.45	0.32	0.00	7.00	4.27	ND	ND
Total recovery	113.63	100.40	93.89	98.54	91.63	89.21	89.66	91.92	87.53

 * Including GI/ingesta

** Additional radioactivity found on skin/fur due to soiling with excreta

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
The majority of the test material was eliminated in faeces with no gender-related differences. Urinary excretion of the low dose of test material ranged from 10 - 16 % with no gender-related differences regardless of the number of doses or routes of exposure. At 168 hours post-dosing, 17 - 34 % of the low dose test material was recovered with the tissues while only ~1 % of the high dose was recovered with the tissues. There were no gender-related differences in the distribution pattern of radioactivity in different tissues of rats.

Executive summary:

This study was conducted to provide information on the absorption, tissue distribution, metabolism and elimination of ¹⁴C-radiolabelled test material following single or multiple oral dosing or a single intravenously administered dose. The study was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 870.7485, OECD 417 and EU Method B.36.

Following dosing with radio-labelled test material, animals were housed individually in glass metabolism cages and urine and faeces collected at 24 hour intervals until the end of the study, 168-hour post-dosing.

No volatile organics and/or ¹⁴CO₂ were exhaled. Radioactivity was determined in the aliquots taken from the collected excreta and tissue samples after oxidisation using a liquid scintillation spectrometer. Equal volume aliquots of urine and faecal homogenates samples from the 0 - 24 hour collection interval were pooled into their respective dose and sex groups and analysed for metabolite(s) by LC/MS/MS. Blood/plasma had low radioactivity and thus were not chemically profiled. The mean total recovery from all groups ranged from 88 to 114 % of the administered dose.

Based on the radioactivity recovered in tissues, urine and metabolites in faeces, a total of about 41 - 55 % of the low oral dose of test material was absorbed from the GI tract. However, about 60 % of the biliary excreted radioactivity recovered in faeces after IV injection was parent test material. Therefore, the absorption calculated for the oral doses is probably underestimated and likely range from 74 - 93 % of the dose administered. Multiple dosing lowered the absorption of the subsequent radiolabelled test material. Absorption of the high oral dose was saturated as only 2 - 5 % of the administered dose was recovered in tissues and urine, and the amounts of metabolites in faeces were negligible.

The majority of the test material was eliminated in faeces with no gender-related differences. The mean faecal recovery of the low dose of test material was 29 - 35 % dose within 24-hours post-dosing, somewhat higher than the 24-hour biliary elimination of 13 - 17 % following IV injection. Faecal elimination of 100 mg/kg was 87 - 93 % of the administered dose during the first 24 hours. The rate of biliary elimination of the IV-dosed test material was linear with an elimination half-life of 53 and 60 hours for male and female rats, respectively.

Urinary excretion of the low dose of test material ranged from 10 - 16 % with no gender-related differences regardless of the number of doses or routes of exposure. Urinary elimination of test material labelled on the other ring was 2.3-fold higher. Less than 1.5 % of the high dose of test material was recovered in urine. Urinary elimination was biphasic with an initial fast elimination half-life between 20 and 35 hours, whereas the slow elimination half-life was between 62 and 110 hours.

At 168 hours post-dosing, 17 - 34 % of the low dose test material was recovered with the tissues while only ~1 % of the high dose was recovered with the tissues. There were no gender-related differences in the distribution pattern of radioactivity in different tissues of rats. The level of radioactivity in tissues between low and high doses was not proportional to the difference between doses and mostly accounted for only 4 to 5-fold increase at the high dose. Multiple dosing reduced levels of the final radiolabelled dose in most tissues by an average of 29 - 50 % when compared with the levels after single oral dose.

A total of five metabolites were found in excreta at levels of >5 % of the administered dose. Most of the radioactivity recovered in faeces was parent test material (51 - 61 % and 84 - 100 % of the recovered radioactivity after IV and oral doses, respectively). Four minor metabolic peaks were found in faeces, two of them eluted before and two after the parent compound based on reverse-phase HPLC separation. A total of 2 metabolites were found in urine of rats dosed orally, and a third metabolite was detected in females dosed IV. Three different peaks, all eluting earlier on the reverse-phase HPLC column, were found in urine of animals dosed with test material labelled on the other ring. The test material is either excreted unchanged (primarily via faeces) or metabolised via cleavage of the acyl urea moiety followed by conjugation with glycine and excretion via urine. The corresponding aniline metabolite is hydroxylated and conjugated with sulfate or glucuronic acid.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2002 - 10 December 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

other: pharmacokinetics and metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

13-C

Species:

rat

Strain:

Fischer 344

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 141 - 151 g
- Fasting period before study: No
- Housing: During acclimation animals were housed two per cage in stainless steel cages and for at least 2 days in individual metabolism cages. The stainless steel cages had wire-mesh floors suspended above catch pans. Cages contained a hanging feeder and a pressure activated nipple type watering system.
Following administration of the test material, animals were housed one per cage in glass Roth-type metabolism cages. The metabolism cages were designed for the separation and collection of urine and faeces as well as expired air. Air was drawn through the metabolism cages at ~500 mL/minute.
- Individual metabolism cages: yes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 12 - 15 times/hour
- Photoperiod: 12-hour light/dark

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous METHOCEL cellulose ether

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Suitable amounts of radiotracer, dissolved in acetone, were added to flasks and the acetone removed by drying under a gentle stream of nitrogen. Crystals of the test material were dissolved in ethanol (≤1% w/w of the dose suspension). The required amounts of an aqueous solution of 0.5 % METHOCEL were then added and the suspensions were stirred at 4 °C for several (2 - 6) days prior to the administration to the animals. The suspension used for multiple dosing was continuously stirred at 4 °C for the duration of the repeated dose administration period.

VEHICLE
- Amount of vehicle (if gavage): Animals were dosed at a target volume of 5 mL dose suspension per kg of body weight.

DOSE CONFIRMATION
The concentration of the test material in the dose suspensions was determined by high performance liquid chromatography (HPLC) with an ultraviolet (UV) detector. Aliquots of dose suspensions were diluted with acetonitrile prior to HPLC analysis. The concentration was quantified using unlabelled standards. Radioactivity of the dose suspensions was determined by liquid scintillation spectrometer (LSS) of aliquots taken from the dosing suspensions.

HOMOGENEITY AND STABILITY OF TEST MATERIAL
LSS analyses of aliquots of the dose suspensions taken from various locations in the containers were used to confirm homogeneity of the dosing suspensions.
The stability in 0.5 % aqueous METHOCEL was determined in the unlabelled repeated dose solution for up to 12 days in a previous study. Oral dose suspensions were used within 10 days of preparation in this study.

Duration and frequency of treatment / exposure:

One group of animals was exposed to a single dose; a second group was exposed to repeated doses for 10 days.

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
for all animals

No. of animals per sex per dose / concentration:

Two female animals were exposed to single doses; 6 female rats were exposed to repeated doses

Control animals:

no

Details on study design:

STUDY DESIGN
- Single Dose: One rat was sacrificed at 3 hours (Cmax) and the other at 24 hours (½ Cmax) post-dosing.
Radioactivity in the brain remained below the levels that could be used for autoradiographic localisation and thus was not pursued further.
- Multiple Doses: Two rats were sacrificed at 3 hours (Cmax), 24 hours (½ Cmax) and 120 hours (~2 t½s) post-dosing.

Details on dosing and sampling:

SPECIMEN COLLECTION
- Urine
All urine (repeated dose animals only) voided during the study was collected in dry-ice cooled traps at 24 hour intervals. The cages were rinsed with water at the time the traps were changed and the rinse was collected. Each urine specimen and urine/cage rinse was weighed, and a weighed aliquot of each sample was analysed for radioactivity by LSS.
- Faeces
Faeces were collected in dry-ice chilled containers at 24-hour intervals. An aqueous homogenate (~25 % w/w) was prepared and weighed aliquots of these homogenates were oxidised (OxiMate 80 Sample Oxidizer, PerkinElmer Life Sciences, Inc., Boston, MA) and quantitated for radioactivity by LSS.

TERMINAL SACRIFICE
All animals were anaesthetised with CO₂ and sacrificed by exsanguination via cardiac puncture. Following sacrifice, the metabolism cages were washed and an aliquot of the final cage wash (FCW) analysed for radioactivity by LSS.

BLOOD/PLASMA/RBC
Blood was obtained at sacrifice via cardiac puncture and radioactivity determined in aliquots of whole blood. Following that, blood was centrifuged to separate plasma from red blood cells and radioactivity determined in both of the fractions by LSS.

TISSUES
The following tissues were collected at sacrifice and analysed:
- Single dose animals: Blood, brain, plasma and RBC
- Repeated dose animals: Adrenal, blood, brain, fat, GI tract (including ingesta), kidney, liver, ovaries, plasma, residual carcass, skin, spleen, RBC and uterus.
The residual carcass, brain (single dose animals only), GI/ingesta, liver and kidney were homogenised (~33 % w/w aqueous homogenate) and a weighed aliquot was oxidised and analysed for radioactivity by LSC. Weighed aliquots of plasma were mixed directly with liquid scintillation fluid and analysed for radioactivity by LSS. Adrenal, samples of perirenal fat, ovaries, blood, spleen and uterus were directly oxidised without homogenisation and analysed for radioactivity by LSS. The skin was removed from the carcass and digested in tetraethylammonium hydroxide (TEAH) and radioactivity determined in aliquots by LSS and used for mass-balance of the administered doses.
Since there was not enough radioactivity in the brains of the single dose animals used for pilot screening, no additional animals were dosed to conduct autoradiography for localisation. Radioactivity in the brain of the rat sacrificed 3 hours (Cmax) after dosing was only 0.00239 μCi/g which was reduced to 0.00198 μCi/g in the animal sacrificed 24 hours (½ Cmax) after dosing. These radioactivity levels in brain were at least a magnitude lower than what has been previously reported to be high enough to obtain a good autoradiographic localisation.

SECTIONING OF BRAIN AND AUTORADIOGRAPHY FOR LOCALISATION
Brains from the repeated dose animals were carefully removed from skull immediately (within 2 - 3 minutes) after the sacrifice and processed for autoradiography. Each brain was placed vertically on the brain stem, covered with powdered dry ice to hasten freezing and maintain anatomical features, and stored at -80 °C until sectioning. One of the brains (3 hours after the last dose, plasma Cmax) was moved to -20 °C for 16 hours before sectioning and sectioned at -20 °C using a Tissue-Tek® II Cryostat (Miles Inc., Elkhart, IN). Transverse sections 16 μm thick were collected every ~500 μm, beginning at the olfactory lobe and progressing through the cerebellum. Sections were thaw-mounted (thawed with finger on the opposite side of glass slide) on electrostatically charged glass slides (Superfrost® Plus, VWR Scientific, West Chester, PA), which had been stored at -20 °C prior to starting sectioning. All glass mounted brain sections were kept in a plastic glass slide holder, sealed with cryogenic tape and stored at -80 °C until autoradiography. All shavings and unused sections of the brain were collected. Slides were mounted on the BioMax TransScreen LE intensifying screen while keeping the screen on dry ice, covered with plastic film, exposed to Kodak BioMax MS Scientific imaging film (Eastman Kodak Co., Rochester, NY) and stored at -80 °C for 51 days. The film was developed and digitised by scanning. The sections were subsequently stained with haematoxylin and eosin (H&E) and used to identify different brain regions. Since all the radioactivity was localised to the blood vessels, no other brain samples (24 and 120 hours after the 10th radiolabeled dose) were processed for autoradiography.

FINAL CAGE WASH
Following the terminal sacrifice of the animals, a final cage wash was performed. The final cage wash was collected and the weight of the sample was determined. A weighed aliquot of the final cage wash was analysed for radioactivity and used for mass-balance of the administered doses.

SAMPLE ANALYSIS
- Tissue Oxidisation
The whole tissue (adrenal, ovaries, spleen, uterus, and aliquots of fat and blood) or a weighed aliquot of the homogenised tissue (carcass, brain, GI/ingesta, liver, kidney, and testes) sample was oxidised using the OxiMate 80 sample oxidiser and analysed for radioactivity by LSS as described below.
Total radioactivity in the brain serially sectioned for autoradiography was determined in the aliquot of the collected shavings and unused sections after homogenisation as described above.
- ¹⁴C Analysis
Radioactivity was quantified in a liquid scintillation spectrometer (Packard Tri-Carb® 2900TR, Packard Bioscience Company, Meriden, CT). Counts per minute (cpm) were corrected for quench and background, and converted to disintegrations per minute (dpm).
The instrument is equipped with sealed ¹⁴C-standards that are counted every 23 hours during the operation to monitor the performance of the LSS.

Statistics:

Descriptive statistics were conducted (i.e., mean ± standard deviation). All calculations in the database were conducted using Microsoft Excel® spreadsheets in full precision mode (15 digits of accuracy). The daily elimination of the administered dose while animals were repeatedly dosed was calculated on the basis of the total body burden (dose remaining to be eliminated). The total body burden was calculated by subtracting the cumulative total dose eliminated from the total cumulative dose administered. Similarly, the cumulative total elimination was calculated by using the total cumulative dose administered and eliminated for each successive day during the course of dosing.

Preliminary studies:

In the singly-dosed animals, radioactivity in the brain was 2.39 nCi/g and 1.98 nCi/g in rats sacrificed 3 and 24 hours post-dosing, respectively, which was much lower than that needed (6 nCi/g) to obtain good autoradiographic localisation within the brain. Concentration of the administered dose was 0.08 %/g (0.11 μg-equivalent/g) in the plasma of the rat sacrificed 3 hours post-dosing which dropped to 0.03 %/g (0.04 μg- equivalent/g) 24 hours post-dosing. Concentration of the administered dose in whole blood and RBCs was 0.06 - 0.07 %/g (0.09 - 0.10 μg-equivalent/g) at 3 hours post-dosing and declined to 0.02 %/g (0.03 μg-equivalent/g) at 24 hours post-dosing. Since not enough radioactivity was found in the brains to allow for determination of localisation through autoradiography, no additional animals were dosed.

Details on distribution in tissues:

At 3, 24 and 120 hours after the final dose, all tissues contained quantifiable radioactivity containing 0.005 % of the administered dose.
A total of 37, 28 and 17 percent of the orally dosed test material remained in tissues 3, 24 and 120 hours after the final radiolabelled dose, respectively. Most of the radioactivity was found in the residual carcass accounting for 16, 14 and 9 percent of the dose. The GI tract of rats sacrificed 3 hours post dosing contained the second highest amount of radioactivity (~10 %), most of which may represent unabsorbed test material remaining in the GI tract. The amount in the GI tract reduced to 4.6 and 2.5 % at 24 and 120 hours post-dosing, respectively.
Distribution of the absorbed dose was in the order of fat >> adrenal = skin > ovaries followed by spleen and liver. This was consistent with the earlier findings previously reported. Distribution of radioactivity to the brain remained low; radioactivity in the brain of the rat sacrificed at Cmax (3 hours after the last dose) was only 7.1 nCi/g, which resulted in reasonably good autoradiographic images showing localisation within the brain being obtained. Distribution of test material derived radioactivity after multiple (10 doses) dosing with radiolabelled test material was only 3-fold higher than what was observed after a single dose, suggesting its low affinity to brain.

- Localisation of the test material derived radioactivity within brain
Brains were step-sectioned beginning at the olfactory lobe (step section #1) to the caudal aspect of the cerebellum/medulla (step section #42) and mounted on glass slides for autoradiography. Following autoradiography, the sections were stained with haematoxylin and eosin for histologic localisation. The brain atlas of Pellegrino et al. (1979) was used for anatomical reference. There was no definitive autoradiographic localisation in the neuropil of any section. Definitive localisation of test material derived radioactivity was found in sections that contained the choroid plexus within the ventricular system of the brain. Sequentially, choroid plexus localisation was first noted in a lateral ventricle in step section #13 and was consistently present in the choroid plexus of the lateral and third ventricles through step section #22. Good autoradiographic localisation resumed with the choroid plexus of the fourth ventricle associated with the medulla and cerebellum in step sections #28 through #39. Localisation within the choroid plexus is consistent with the increased permeability of the microvasculature of the choroid plexus.
Lesser or faint autoradioactive localisation was found inconsistently and relatively infrequently on the surface of brain or between opposing brain structures. This localisation was considered to be associated with the meninges (arachnoid and pia mater), particularly with blood vessels at these sites. The pattern of localisation of test material derived radioactivity in brain indicates that the test material does not cross the blood-brain barrier and thus, probably does not directly lead to convulsions seen in the reproductive toxicity study.

Details on excretion:

Urinary excretion of the test material ranged between ~3 and 4 percent of the remaining dose in the body during the course of the dosing (day 1 through day 10). Daily elimination was slightly higher (3.5 - 4.3 % of the body burden) during the early days of dosing (day 1 through day 5) compared to the last 5 days, which was approximately 2.5 - 3.0 % of the body burden. A total of about 12 percent of the administered dose was eliminated in urine. This was similar to the reported urinary excretion of the test material after a single oral dose or multiple (14 daily doses) non-radiolabelled doses following by a single oral radiolabelled dose reported in an earlier study.
A majority of the orally dosed test material was eliminated in faeces, accounting for 63 % of the total doses. This was similar to what was observed after a single oral dose (60 % of the administered dose) or multiple (14 daily doses) non-radiolabelled doses followed by a single oral radiolabelled dose (56 % of the administered dose) reported in the earlier study.
Similar to the pattern of elimination observed for the urinary excretion, a much higher level of radioactivity was eliminated in faeces during the first 4 days of dosing accounting for 20 to 32 percent of the body burden. During the last 6 days of dosing, faecal elimination was lower and ranged between 15 and 19 percent of the body burden. A total of 75 % of the administered dose was eliminated from the body during the course of the study. This was again consistent with the earlier findings that the excretion of the radioactivity was 71 and 67 % after single and multiple dosing, respectively.
The lowering in the urinary and faecal elimination with successive doses represents two factors. One is the increase in the total body burden (dose remained inside the body, accumulation) with each successive dose; there was an increase in the total body burden ranging from 10 μCi 24 hours after the first dose to 48 μCi after 10th dose. The other factor that resulted in the decline of the radioactivity elimination and increase in the total body burden, although reached almost a steady-state elimination of 18 - 19 % between 7 and 10 days of dosing and accumulation of 43 - 48 μCi during the last three days of dosing, was the lack of reaching a true steady-state level. This was consistent with the long elimination half- life of the test material seen previously in female Fischer 344 rats of 61 - 86 hours. The course of 10 days of dosing covered only 3 - 4 half-lives, whereas about 7 half-lives are needed to achieve true steady-state exposure levels with only about 1 % perturbation. The fluctuation in the elimination and tissue accumulation is consistent with the expected achievement of 88 - 94 % of the steady-state levels from 3 - 4 half-lives.

The concentration of the test material in the dose suspension used for the repeated dose animals was 0.202 mg/g METHOCEL and the concentration of radioactivity was 14.1 μCi/g dose suspension.

The mean daily dose administered to each rat was 10.6 ± 0.1 μCi (71.6 ± 0.5 μCi/kg) and 0.15 ± 0.00 mg (1.03 ± 0.01 mg/kg). Each animal received a total cumulative dose of 106.3 ± 1.0 μCi and 1.52 ± 0.01 mg during the course of dosing (10 days).

The mean recovery of the administered dose was 92 ± 3 % ranging from 88 to 96 %.

Conclusions:

Distribution of the radioactivity within the brain was focal and exclusively localised in blood vessels on the surface of the brain or in the choroid plexus of the ventricles, none of the test material derived radioactivity crossed the blood brain barrier.

Executive summary:

The purpose of this study was to determine the time-course tissue distribution and localisation of radioactivity within different regions of the brain after multiple oral doses of radiolabelled test material in rats. Determination of localisation of test material derived radioactivity in different regions of the brain was an attempt to find a relationship, if any, between the accumulation of the radioactivity in specific structures in brain and the cause of convulsions seen in rat pups observed in the high-dose groups of previous reproductive toxicity studies.

The study was conducted in accordance with the standardised guidelines OECD 417, EU Method B.36 and US EPA OPPTS 870.7485 under GLP conditions.

Six female Fischer 344 rats were administered radiolabelled test material (1.03 ± 0.01 mg/kg/day; (71.6 ± 0.5 μCi/kg/day) for ten days via oral gavage of a METHOCEL suspension of the test material. Rats were housed individually in glass metabolism cages following the first oral dose and urine and faeces were collected at 24 hour intervals until the end of the study. Two rats were sacrificed 3, 24 and 120 hours after the final (10th) oral dose. Blood was collected via cardiac puncture under anaesthesia. The brain was carefully removed from each rat immediately after the sacrifice and frozen on powdered dry ice. Radioactivity was determined directly in the aliquots taken from the collected urine, radioactivity in faeces and tissues was determined after oxidisation using a liquid scintillation spectrometer. Brains were stored at -80 °C until analysis. One of the brains (3 hours after the last dose, plasma Cmax) was brought to -20 °C for 16 hours before serial transverse sectioning (16 μm) beginning at the olfactory lobe and progressing through the cerebellum using a cryomicrotome at -20 °C. One section every ~500 μm apart was thaw-mounted on electrostatically charged glass slides and localisation of test material derived radioactivity within the brain was determined by conventional autoradiography using sensitive film and an intensifying screen.

Each rat received a mean daily dose of ~11 μCi (~0.15 mg), the cumulative dose over the ten days was 106 ± 1 μCi (1.52 ± 0.01 mg). The mean recovery of the administered doses was 92 ± 3 %. Daily urinary excretion of the 14C-test material derived radioactivity was between 3 and 4 % while animals were still receiving radiolabelled doses, which resulted in the cumulative total urinary elimination of about 12 % of the dose. Most of the administered radioactivity was eliminated in faeces, the daily faecal elimination during the course of the dosing was 15 - 32 % totalling to the cumulative faecal elimination of 63 % of the dose. It appeared that most of the administered doses were absorbed from the GI tract before its elimination into faeces through bile. A total of 37, 28 and 17 percent of the dose remained in tissues 3, 24 and 120 hours after the final radiolabelled dose. Distribution of the absorbed radioactivity to tissue was in the order of fat >> adrenal = skin > ovaries followed by spleen and liver. Distribution of radioactivity to the brain remained low; radioactivity in the brain of the rat sacrificed at Cmax (3 hours after the last dose) was only 7.1 nCi/g.

A true steady-state level was not reached during the course of 10 daily doses, which was expected from the reported long half-life of the test material in rats (61 - 86 hours), although a close to steady-state level of elimination was reached with 6 - 12 % variation at the end of the dosing period. Distribution of the radioactivity within the brain was focal and exclusively localised in blood vessels on the surface of the brain or in the choroid plexus of the ventricles, none of the test material derived radioactivity crossed the blood brain barrier.

Description of key information

ADME data suggests that noviflumuron is readily absorbed at low dose (65%), although absorption rapidly becomes saturated at higher doses.  Bioaccumulation is potentially high, with long terminal have lives, and as illustrated by tissue concentrations increasing with repeat doses. Primary route of excretion is biliary, and ultimately via the feces with minor amounts being excreted via the urine. The test material is concentrated in the milk of lactating animals. The test material is either excreted unchanged (primarily via faeces) or metabolised via cleavage of the acyl urea moiety followed by conjugation with glycine and excretion via urine. The corresponding aniline metabolite is hydroxylated and conjugated with sulphate or glucuronic acid.

Dermal absorption of 10% was estimated on the basis of the physical chemical properties.  Inhalation absorption was considered to be 100% in the absence of any inhalation absorption data. Tissue accumulation data indicated a high potential for bioaccumulation.

Key value for chemical safety assessment

Bioaccumulation potential:

high bioaccumulation potential

Absorption rate - oral (%):

65

Absorption rate - dermal (%):

10

Absorption rate - inhalation (%):

100

Additional information

Three ADME studies have been conducted for noviflumuron: Saghir et al., 2002; Saghir et al., 2003; and Saghir et al., 2004. In Saghir et al., 2002, groups of animals received single oral doses of 10, 100 or 250 mg/kg of fluorodichlorophenyl labelled material.  In Saghir et al., (2003) groups of animals received either 1 or 100 mg/kg bw of fluorodichlorophenyl labelled test material, or 1 mg/kg bw difluorobenzoyl ring labelled test material. Another group received 14 daily 1 mg/kg bw doses of unlabelled test material and 1 mg/kg bw of fluorodichlorophenyl ring labelled test material on day 15. A fifth group received a 1 mg/kg bw IV dose of fluorodichlorophenyl ring labelled test material. In Saghir et al., (2004) six female Fischer 344 rats were administered fluorodichlorophenyl radiolabelled test material (1.03 ± 0.01 mg/kg/day; (71.6 ± 0.5 μCi/kg/day) for ten days via oral gavage of a METHOCEL suspension of the test material. 

 

Absorption

Oral absorption

In Saghir et al., (2003), based on the radioactivity recovered in tissues, urine and metabolites in faeces, a total of about 41 - 55% of the low oral dose of test material was absorbed from the GI tract. However, about 60% of the biliary excreted radioactivity recovered in faeces after IV injection was parent test material. Therefore, the absorption calculated for the oral doses is probably underestimated and likely range from 74 - 93% of the dose administered. Multiple dosing lowered the absorption of the subsequent radiolabelled test material. Absorption of the high oral dose was saturated as only 2 - 5% of the administered dose was recovered in tissues and urine, and the amounts of metabolites in faeces were negligible (Saghir et al., 2003).

In Saghiret al., (2002) the total urinary excretion through urine by 72 hours post dosing was identical at 100 and 250 mg/kg bw. The similar levels of recovery and the drop in urinary clearance observed at these dose levels were indications of saturation of absorption and/or metabolism in animals. Similarly, non-proportionalities in AUC and Cmax and higher faecal elimination suggest saturation of absorption from the GI tract that caused a decrease in bioavailability at 100 and 250 mg/kg (Saghiret al., 2002).

Based on Saghir et al., (2003), oral absorption is conservatively considered to be 65%.

 

Dermal absorption

No dermal absorption data is available. On the basis of a molecular weight > 500 and a Kow >4, dermal absorption is taken as 10% (ECHA Guidance R.7)

Inhalation absorption

In the absence of data inhalation absorption is considered to be 100% (ECHA Guidance R.7).

Distribution

In Saghir et al., (2003) at 168 hours post-dosing (low dose), 17 - 34% of the low dose test material was recovered with the tissues while only ~1% of the high dose was recovered with the tissues. There were no gender-related differences in the distribution pattern of radioactivity in different tissues of rats. The level of radioactivity in tissues between low and high doses was not proportional to the difference between doses and mostly accounted for only 4 to 5-fold increase at the high dose. Multiple dosing with cold material prior to radiolabelled dose reduced levels of the final radiolabelled dose in most tissues by an average of 29 - 50% when compared with the levels after single oral dose (Saghir et al., 2003).

In Saghir et al. (2004), a total of 37, 28 and 17 percent of the dose remained in tissues 3, 24 and 120 hours after the final radiolabelled dose. Distribution of the absorbed radioactivity to tissue was in the order of fat >> adrenal = skin > ovaries followed by spleen and liver. Distribution of radioactivity to the brain remained low; radioactivity in the brain of the rat sacrificed at Cmax (3 hours after the last dose) was only 7.1 nCi/g (Saghir et al., 2004). A true steady-state level was not reached during the course of 10 daily doses, which was expected from the reported long half-life of the test material in rats (61 - 86 hours), although a close to steady-state level of elimination was reached with 6 - 12% variation at the end of the dosing period. Distribution of the radioactivity within the brain was focal and exclusively localised in blood vessels on the surface of the brain or in the choroid plexus of the ventricles, none of the test material derived radioactivity crossed the blood brain barrier (Saghir et al., 2004).

There was preferential partition into milk of lactating females. Exposure of foetuses through the placenta is limited and the majority of exposure occurs postnatally through nursing. The systemic exposure of neonates through milk surpassed the systemic exposure to non-lactating dams by approximately 3-fold

(Saghir & Markham, 2005).

 

Metabolism

In Saghir et al. (2003), a total of five metabolites were found in excreta at levels of >5% of the administered dose. Most of the radioactivity recovered in faeces was parent test material (51 - 61% and 84 - 100% of the recovered radioactivity after IV and oral doses, respectively). Four minor metabolic peaks were found in faeces, two of them eluted before and two after the parent compound based on reverse-phase HPLC separation. A total of 2 metabolites were found in urine of rats dosed orally, and a third metabolite was detected in females dosed IV. Three different peaks, all eluting earlier on the reverse-phase HPLC column, were found in urine of animals dosed with test material labelled on the other ring. The test material is either excreted unchanged (primarily via faeces) or metabolised via cleavage of the acyl urea moiety followed by conjugation with glycine and excretion via urine. The corresponding aniline metabolite is hydroxylated and conjugated with sulphate or glucuronic acid (Saghir et al., 2003).

In Saghiret al. (2002), after single oral doses of 10, 100 or 250 mg/kg: Three urinary metabolites were tentatively identified using HPLC/LC-MS analysis; these metabolites include the glucuronide and sulphate conjugates of hexafluoroalkoxy-fluorodichloroaniline, as well as a mercapturic acid conjugate of the parent compound. No differences were observed in the profile of urinary metabolites between sexes or dose levels (Saghir et al., 2002).

Excretion

In Saghir et al., (2002), after single oral doses of 10, 100 or 250 mg/kg: The primary route of excretion was determined to be through faecal elimination. Within 72 hours, 53 - 90% of the administered dose was recovered in the faeces. Faecal elimination was rapid in males with 88-95% of the eliminated radioactivity recovered within 24 hours. Less so in females: In females, 54 -77% of the total faecal elimination of radioactivity was recovered within 24 hours and 77 - 96% within 48 hours. Males eliminated 53 - 69% and females eliminated 58 - 90% of dose in the faeces. At all dose levels, only 0.7 - 5.0% was recovered in the urine (Saghir et al., 2002).

In Saghir et al., (2003) groups of animals received either 1 or 100 mg/kg bw of fluorodichlorophenyl ring labelled test material, or 1 mg/kg bw difluorobenzoyl ring labelled test material. Another group received 14 daily 1 mg/kg bw doses of unlabelled test material and 1 mg/kg bw of fluorodichlorophenyl ring labelled test material on day 15. A fifth group received a 1 mg/kg bw IV dose of fluorodichlorophenyl ring labelled test material. 

The majority of the test material was eliminated in faeces with no gender-related differences. The mean faecal recovery of the low dose of test material was 29 - 35% dose within 24-hours post-dosing, somewhat higher than the 24-hour biliary elimination of 13 - 17% following IV injection. Faecal elimination of 100 mg/kg was 87 - 93% of the administered dose during the first 24 hours. The rate of biliary elimination of the IV-dosed test material was linear with an elimination half-life of 53 and 60 hours for male and female rats, respectively.

Urinary excretion of the low dose of test material ranged from 10 - 16% with no gender-related differences regardless of the number of doses or routes of exposure. (Urinary elimination of test material labelled on Difluorobenzoyl ring labelled was 2.3-fold higher). Less than 1.5% of the high dose of test material was recovered in urine. Urinary elimination was biphasic with an initial fast elimination half-life between 20 and 35 hours, whereas the slow elimination half-life was between 62 and 110 hours. (Saghir et al., 2003). Urinary elimination was biphasic with an initial fast elimination half-life between 20 and 35 hours, whereas the slow elimination half-life was between 62 and 110 hours (Saghir et al., 2003).

References

Saghir SA & Markham DA (2005) XDE-007: Mechanistic Study to Evaluate Possible Cause of Toxicity Differences Among Species. Study report no. 021125. The Dow Chemical Company, Midland, Michigan, USA. GLP. Not published

Saghir SA, Mendrala AL & Rick DL (2002) XDE-007: Limited Pharmacokinetics and Metabolism of 14C-Labelled XDE-007 Following a Single Oral Administration in Fischer 344 Rats. Study Report no. 011151 The Dow Chemical Company, Michigan, USA. GLP. Not published.

Saghir SA, Mendrala AL & Clark AJ (2003) XDE-007: Pharmacokinetics and Metabolism in Fischer 344 Rats (Part A). Study report no. 021110. The Dow Chemical Company, Michigan, USAGLP. Not published.

Saghir SA, Johnson KA, Mendrala AL & Clark AJ (2004) XDE-007: Pharmacokinetics and metabolism in Fischer 344 rats (Part B) Study no. 021110B The Dow Chemical Company, Michigan, USA. GLP. Not published.