benzovindiflupyr (ISO); N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

Endpoint summary

Additional information

Absorption

The oral absorption of total radioactivity was estimated following a single low or high oral gavage dose of SYN545192 (1 or 40 mg/kg) to bile duct cannulated male and female rats. Oral absorption was estimated as the radioactivity present in urine, bile, cage wash, faeces and carcass up to 48 hours post dose by which time the majority of the administered radioactivity had been excreted (97 and 89% following 1 mg/kg and 90 and 86% following 40 mg/kg in males and females, respectively). Absorption was similar in male and female rats following both doses. Absorption following 1 mg/kg (low dose) was estimated to be 81% in males and 79% in females. Following the 40 mg/kg (high dose), absorption was estimated to be 61 and 62% in males and females, respectively.

Excretion

Irrespective of dose or sex, following a single oral dose of 1 or 40 mg/kg of SYN545192 radioactivity was distributed throughout the body and was rapidly eliminated with the majority being excreted within the first 72 hours post dose (95 and 91% following the low dose and 97 and 91% following the high dose in males and females, respectively). Following both doses the predominant route of elimination wasviathe faeces. Following a single oral dose of 1 mg/kg, eliminationviathe faeces accounted for 84 and 90% of the administered dose and urinary excretion accounted for 12 and 6 % of the administered dose in males and females, respectively. Following a single oral dose of 40 mg/kg, the faeces accounted for 93 and 90% of the administered dose in males and females, respectively, with urinary excretion accounting for 7% in both sexes. 

With the high recovery of radioactivity in faeces, biliary elimination was shown to be an important route of excretion following both doses in both sexes. In the 48 hours following dosing 76 and 69% of the administered dose was excreted via bile following 1 mg/kg and 47 and 57% of the administered dose was excreted via bile following 40 mg/kg in males and females, respectively.

In a preliminary study (QWBA), using both radiolabels, and in the excretion and distribution study, using pyrazole labelled SYN545192, radioactivity was measured in expired air over the first 24 hours post dose and shown to be negligible (<0.1% of administered dose) in both sexes. This was consistent with the generally high recoveries of dose and the anticipated metabolically stable location of the radiolabels in the molecule.

Distribution

In the preliminary QWBA study using both radiolabels of SYN545192 total radioactivity was extensively distributed throughout the body by the first sampling time of 5 hours in males and 1 hour in females and had declined markedly in both sexes by 72 hours post dose. There were no clear differences in tissue distribution profiles between the labels nor were any pronounced sex or dose differences apparent. The highest tissue concentrations were present in the Harderian gland and liver with lower concentrations in the adrenal gland, brown fat and kidney.

The excretion and distribution study demonstrated that following both doses in both sexes the residues of radioactivity at 7 days post dose were very low in the tissues and carcass with only 1.8 and 1.0% of the dose remaining following the 1 mg/kg dose of SYN545192 and 1.4 and 0.8% remaining following the 40 mg/kg dose in males and females, respectively.

Seven days following administration of the low dose to male rats, radioactivity was detected in the blood and plasma at a concentration of 0.033 and 0.040 µg equiv/g, respectively. Mean tissue concentrations in the kidney and liver were 0.055 µg equiv/g and 0.046 µg equiv/g, respectively. However, concentrations of radioactivity in all other tissues were below that of the blood concentration. Radioactivity in the blood and plasma of female rats was 0.004 and 0.003 µg equiv/g, respectively. The highest mean tissue concentrations were also present in the kidney and liver, with a common mean of 0.016 µg equiv/g. Lower concentrations of radioactivity were also found in the thyroid, adrenals and renal fat ranging between 0.013-0.015 µg equiv/g. All other tissues, except bone mineral and brain were also above that of the blood concentration.

Seven days following administration of the high dose to male rats, the concentration of radioactivity in the blood and plasma was 0.53 and 0.63 µg equiv/g, respectively. The highest mean tissue concentrations were present in the kidney and liver at 1.48 and 1.30 µg equiv/g, respectively. Progressively lower concentrations, but above those in blood, were present in the thyroid, heart, adrenal glands, pancreas, lungs and spleen. Concentrations of radioactivity in the remaining tissues were below that of the blood concentration or were not reliably detected. The concentration of radioactivity in the blood and plasma of female rats was 0.23 and 0.15 µg equiv/g, respectively. The highest mean tissue concentrations were present in the kidney and the liver at 0.82 and 0.76 µg equiv/g, respectively. Progressively lower concentrations, but above those in blood, were present in the renal fat, heart, pancreas, lungs, spleen and ovaries. Concentrations of radioactivity in the remaining tissues were below that of the blood concentration or were not reliably detected.

Pharmacokinetics

Following a single oral dose of SYN545192 to male and female rats, peak plasma concentrations of radioactivity were reached after approximately 2 - 4 hours and 6 - 24 hours following doses of 1 and 40 mg/kg, respectively. Following the low dose the terminal phase half-life was 55 hours in males and 28 hours in females and following the high dose was 30 hours and 33 hours in males and females, respectively. Systemic exposure to total radioactivity was comparable between plasma and whole blood but there was evidence to suggest a greater systemic exposure to total radioactivity in males compared to females. Increases in exposure with respect to Cmax and AUC were generally less than proportional with the increase in dose but there appeared to be a trend towards broad dose proportionality in AUC estimates in females.

Tissue depletion following a single oral dose

A single oral dose of 1 mg or 40 mg/kg of SYN545192 was administered to male and female rats to investigate the tissue distribution of radioactivity. At intervals over a period of 6 days after dosing, the rats were killed in groups of 3 per sex and residual radioactivity was measured in selected tissues/organs and the remaining carcasses. Tissue distribution was extensive throughout and generally similar between the sexes following both doses. Following an initial rapid decline terminal phase half-life estimates for tissue depletion appeared slightly longer in male animals than in female animals following the low dose. However, this trend was not apparent following the high dose and, therefore, may not reflect real physiological differences between the sexes. Collectively tissue concentrations of radioactivity were highest at the first sampling time point and progressively declined thereafter with terminal phase half-lives ranging between 1.4 and 8.8 days. The calculation of half lives for some tissues was made difficult because of low and variable tissue concentrations measured over the course of the study.

The highest concentration of radioactivity following both doses was present in the liver of both sexes with liver, kidney and adrenal concentrations remaining the highest throughout the course of the study. The total residues in tissues and carcass at the end of the study accounted for 2.4% of the dose in males and 2.0% in females following the low dose and for just 1.1% and 3.8% following the high dose in males and females, respectively.

Tissue depletion following repeated oral dosing

Daily oral doses of 1 mg/kg SYN545192 was administered to male rats for 14 days to determine the extent of accumulation of radioactivity in tissues and the remaining carcasses and its subsequent elimination. Radioactivity was well distributed into the tissues and the concentration generally increased during the period of dosing and appeared to be approaching steady state concentrations by the end of the 14 day dosing period. Following the cessation of dosing, all tissue concentrations steadily declined.

Tissue concentrations of radioactivity were highest in the liver and kidney consistent with both biliary and urinary elimination of [¹⁴C]-SYN545192 and its metabolites. Following the liver and kidney, the adrenals and thyroid had the greatest concentrations of radioactivity with residues in all other tissues being generally below plasma concentrations until around 10 days after the last dose on Day 14. Thereafter, concentrations were generally above those in plasma and by the final sampling time (63 days after the dose on Day 14) concentrations were still measurable in most tissues but, were approaching the limit of reliable measurement. The total tissue and carcass residues at the final sampling time accounted for less than 2.5% of the total radiolabelled dose administered.

As was observed following a single dose the terminal phase half-life for tissue depletion was variable reflecting the low concentrations at or around the limit of reliable measurement for several tissues. As a result the shortest estimate was obtained in the plasma and the longest in the testes being 2.5 and 69 days, respectively.

Biotransformation

SYN545192 was extensively metabolised in rat giving rise to at least 8 types of metabolite (e.g.desmethyl, hydroxy, dihydroxy, desmethyl hydroxy, desmethyl dihydroxy, ring-open, glucuronide conjugate, sulphate conjugate) with the potential for multiple isomers within most types. The majority of the administered radioactivity (70-85% dose) was identified following a single oral dose of SYN545192. The major metabolites were identified as SYN546041, SYN546360, SYN546643, SYN546645 and SYN546619. Other identified components included SYN546039, SYN546042, SYN546708 and SYN546644. Glucuronide and, in some cases, sulphate conjugates of these metabolites were also present. While some quantitative differences were observed between males and females, SYN546041 and SYN546360 together accounted for a major proportion of the dose (35-60% dose). No significant differences were observed following a single dose at 40 mg/kg or 1 mg/kg or repeat daily dosing at 1 mg/kg/day. Minor differences were observed between males and females as indicated by the presence of ring-open metabolites, SYN546634, SYN546706 and SYN546707 primarily in males and the presence of a sulphate conjugate of SYN546042 in females only. There was little evidence to indicate cleavage of SYN545192 between the pyrazole and phenyl moieties with possible metabolites present at <2% of the dose in urine only.

The biotransformation proceeded by:

• Formation of SYN546206 by N-demethylation of SYN545192 Hydroxylation and demethylation to give the major metabolite SYN546041Hydroxylation and demethylation to give the major metabolite SYN546041
• Hydroxylation of SYN545192 to give the major phenolic metabolite SYN546360
• Hydroxylation of both SYN545192 and SYN546206 to give the metabolites SYN546039, SYN546360, SYN546040, SYN546042 and SYN546708
• Further hydroxylation to give dihydroxylated metabolites of both SYN545192 and SYN546206 (e.g. SYN546619, SYN546644, SYN546645 and SYN546643)
• Opening of the bicyclo moiety of both SYN545192 and SYN546206 to give metabolites SYN546634, SYN546706 and SYN546707
• Glucuronic acid conjugation and some sulphate conjugation

Details on the codes used in assignment of metabolites and corresponding structures are provided as an attachment in the endpoint study record for Report No. 31096 (Green & MacDonald, 2011).

Discussion on bioaccumulation potential result:

Biliary elimination

After a single oral dose of [pyrazole‑5‑¹⁴C]‑SYN545192 at least 79% of the 1 mg/kg dose and at least 60% of the 40 mg/kg dose was absorbed.  Irrespective of dose or sex, the radioactivity was fairly rapidly and extensively eliminated, predominantly via the bile.  By two days after dosing carcass residues represented 6.1% of the dose or less.

Excretion and Tissue Distribution

Irrespective of dose or sex, a single oral dose of 1 or 40 mg [pyrazole‑5‑¹⁴C]‑SYN545192/kg was rapidly and extensively eliminated with the predominant route of elimination being via the faeces. 

 At both doses, residues of radioactivity were relatively low in blood and tissues by 7 days post dose, but remained detectable in both sexes. Tissue distribution was also similar in both sexes at both doses, with the highest concentrations in the organs of excretion (kidneys and liver). These findings were consistent with the extensive excretion of the administered dose.

Tissue Depletion following Single Oral Dose

Irrespective of dose or sex, the tissue distribution of radioactivity was extensive following a single oral dose of 1 or 40 mg [¹⁴C]‑SYN545192/kg to rats. Tissue concentrations of radioactivity were highest at the first sampling time (4 hours post 1 mg/kg and 24 hours post 40 mg/kg) and progressively declined thereafter with elimination half lives of between 1.4 and 8.7 days. By 144 hours post dose most tissue concentrations were still reliably detectable but were low, with total tissue and carcass residues accounting for less than 3.8% of the dose. The high concentrations of radioactivity in the gastrointestinal tract and its contents were consistent with the established biliary elimination and faecal excretion of SYN545192 and its metabolites.

Tissue Distribution and Elimination following Repeated Daily Oral Adminstration

Following repeated daily oral administration of 1 mg [¹⁴C]-SYN545192/kg to male rats, tissue distribution of radioactivity was extensive and most tissue concentrations appeared to be approaching steady state concentrations after 14 doses. Tissue concentrations of radioactivity were highest in the liver followed by the kidney during dosing with all tissue concentrations declining following the cessation of dosing. By the final sampling time, concentrations were measurable in most tissues, but were approaching the limit of reliable measurement. The total tissue and carcass residues at the final sampling time accounted for less than 0.2% of the total radiolabelled dose administered. The terminal half-lives for tissue depletion were variable reflecting the low concentrations at or around the limit of reliable measurement for several tissues and ranged from 2.5 days for plasma to 69.1 days for the testes.

QWBA

There were no clear differences in tissue distribution profiles between the [pyrazole‑5‑¹⁴C]‑ or [phenyl-U-¹⁴C]‑SYN545192, nor were any pronounced sex or dose differences apparent. Total radioactivity was extensively distributed throughout the body with most tissue concentrations exceeding that in blood throughout the study. Tissue concentrations of radioactivity were highest at the first termination time (5 hours in males or 1 hour in females following the 1 mg/kg dose and 5 hours for both sexes following the 40 mg/kg dose) and had declined markedly by 72 hours post dose. In general, the highest tissue concentrations were present in the Harderian gland and liver with lower concentrations in the adrenal gland, brown fat and kidney.

 Since, expired air accounted for only 0.01% and 0.03% of a 1 mg/kg dose of [pyrazole‑5‑¹⁴C]‑ or [phenyl-U-¹⁴C]‑SYN545192, respectively, both positions of radiolabelling are considered to be metabolically stable with respect to this route of excretion, with no volatile metabolites produced.

Investigation of the Nature and Identity of Radiolabelled Metabolites Present in Plasma, Urine, Faeces and Bile

This study has shown that SYN545192 was extensively metabolised in rat giving rise to at least 8 types of metabolite(e.g.desmethyl, hydroxy, dihydroxy, desmethyl hydroxy, desmethyl dihydroxy, ring-open, glucuronide conjugate, sulphate conjugate) with the potential for multiple isomers within most types. The majority of the administered radioactivity (70-85% dose) was identified following a single oral dose of SYN545192. The major metabolites were identified as SYN546041, SYN546360, SYN546643, SYN546645 and SYN546619. Other identified components included SYN546039, SYN546042, SYN546708 and SYN546644. Glucuronide and, in some cases, sulphate conjugates of these metabolites were also present. While some quantitative differences were observed between males and females, SYN546041 and SYN546360 together accounted for a major proportion of the dose (35-60% dose). No significant differences were observedfollowing a single dose at 40 mg/kg or 1 mg/kg or repeat daily dosing at 1 mg/kg/day. Minor differences were observed between males and females as indicated by the presence of ring-open metabolites, SYN546634, SYN546706 and SYN546707 primarily in males and the presence of a sulphate conjugate of SYN546042 in females only. There was little evidence to indicate cleavage of SYN545192 between the pyrazole and phenyl moieties with possible metabolites present at <2% dose in urine only.

The biotransformation proceeded by:

·        Formation of SYN546206 by N-demethylation of SYN545192

·        Hydroxylation and demethylation to give the major metabolite SYN546041

·        Hydroxylation of SYN545192 to give the major phenolic metabolite SYN546360

·        Hydroxylation of both SYN545192 and SYN546206 to give the metabolites SYN546039, SYN546360, SYN546040, SYN546042 and SYN546708

·        Further hydroxylation to give dihydroxylated metabolites of both SYN545192 and SYN546206 (e.g.SYN546619, SYN546644, SYN546645 and SYN546643)

·        Opening of the bicyclo moiety of both SYN545192 and SYN546206 to give metabolites SYN546634, SYN546706 and SYN546707

·        Glucuronic acid conjugation and some sulphate conjugation

Details on the codes used in assignment of metabolites and corresponding structures are provided as an attachment in the endpoint study record for Report No. 31096 (Green & MacDonald, 2011).