Composition and rodenticidal bait comprising flocoumafen, and process for controlling target rodent pests

Abstract

Disclosed is a composition including flocoumafen and an amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.4, such that the ratio of this amount to the amount of flocoumafen in the composition is less than 10%, the enantiomer E.sub.4 being present with the exclusion of a racemic mixture, the enantiomer E.sub.4 having, by chromatographic analysis of flocoumafen, a retention time t.sub.4 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4; t.sub.1, t.sub.2 and t.sub.3 representing the retention times of the configurational stereoisomers of flocoumafen different from the enantiomer E.sub.4, the chromatographic analysis being performed at a temperature of 23.5 C.

Claims

1. Composition comprising flocoumafen, wherein; said composition comprising flocoumafen either includes: a non-zero amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.4, said enantiomer E.sub.4 being one of the configurational stereoisomers of one of the two diastereoisomers, said diastereoisomer D.sub.1,4, of flocoumafen, such that the ratio of this amount to the amount of flocoumafen in the composition is less than 10%, said enantiomer E.sub.4 having, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen, a retention time t.sub.4 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4; t.sub.1, t.sub.2 and t.sub.3 representing the retention times of the configurational stereoisomers of flocoumafen different from said enantiomer E.sub.4; wherein the amount of said enantiomer E.sub.4 in the composition is different from the amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.1, said enantiomer E.sub.1 being the other configurational stereoisomer of said diastereoisomer D.sub.1,4, different from said enantiomer E.sub.4 and having, by chromatographic analysis of a flocoumafen composition under the conditions mentioned below, a retention time t.sub.1 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, or; alternatively said composition comprising flocoumafen comprises a non-zero amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.1, said enantiomer E.sub.1 having, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen, under the conditions mentioned below, a retention time t.sub.1 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, said enantiomer E.sub.1 being one of the configurational stereoisomers of one of the two diastereoisomers, said diastereoisomer D.sub.1,4 of flocoumafen, said enantiomer E.sub.1 being different from a configurational stereoisomer of flocoumafen, named enantiomer E.sub.4, said enantiomer E.sub.4 being the other configurational stereoisomer of said diastereoisomer D.sub.1,4 said enantiomer E.sub.4 having, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen, a retention time t.sub.4 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, said composition comprising flocoumafen being free of said enantiomer E.sub.4; the chromatographic analysis being performed at a temperature of 23.5 C. and under the conditions below: on a high-pressure liquid chromatography column of dimensions 1502 mm, and comprising a chiral stationary phase constituted of particles of tris(4-chloro-3-methylphenyl carbamate) cellulose, said particles having a mean size of 3 m and having a mean pore size of 1000 ; using, as liquid mobile phase, a mixture formed from acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with an AB volume ratio of 92/8 and with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL/minute; by injection into the chromatography column of a volume of 1 L of flocoumafen composition at a concentration of 1 g of flocoumafen per millilitre of acetonitrile.

2. Composition according to claim 1, comprising an amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.2, which has, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under the abovementioned conditions, a retention time t.sub.2 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, the amount of said enantiomer E.sub.2 being such that the ratio of this amount to the amount of flocoumafen in the composition is less than 10%.

3. Composition according to claim 1, comprising an amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.3, which has, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under the abovementioned conditions, a retention time t.sub.3 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, the amount of said enantiomer E.sub.3 being such that the ratio of this amount to the amount of flocoumafen in the composition is less than 10%.

4. Composition according to claim 1, comprising an amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.1, which has, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under the abovementioned conditions, a retention time t.sub.1 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, the ratio of the amount of said enantiomer E.sub.1 in the composition to the amount of flocoumafen in the composition being between 30% and 100%.

5. Rodenticidal bait comprising a composition according to claim 1 and at least one excipient that is edible for target rodent pests.

6. Bait according to claim 5, wherein the edible excipient comprises at least one food chosen from the group formed from cereal seeds, cereal seed meals, cereal seed flours, cereal seed flakes, cereal bran and non-cereal seeds.

7. Bait according to claim 5, comprising a mass amount of flocoumafen such that the ratio of this mass amount of flocoumafen to the mass amount of rodenticidal bait is less than 200 ppm.

8. Process for controlling target rodent pests, in which there is spread an amount of rodenticidal bait comprising flocoumafen, said bait comprising: at least one excipient that is edible for target rodent pests, wherein the rodenticidal bait comprises either; a non zero amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.4, said enantiomer E.sub.4 being one of the configurational stereoisomers of one of the two diastereoisomers, said diastereoisomer D.sub.1,4, of flocoumafen, such that the ratio of this amount to the amount of flocoumafen in the composition is less than 10%, said enantiomer E.sub.4 having, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen, a retention time t.sub.4 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4; t.sub.1, t.sub.2 and t.sub.3 representing the retention times of the configurational stereoisomers of flocoumafen different from said stereoisomer E.sub.4; characterized in that wherein the amount of said enantiomer E.sub.4 in the composition is different from the amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.1, said enantiomer E.sub.1 being the other configurational stereoisomer of said diastereoisomer D.sub.1,4, different of said enantiomer E.sub.4 and having, by chromatographic analysis of a flocoumafen composition under the abovementioned conditions, a retention time t.sub.1 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, or; alternatively the rodenticidal bait comprises (may comprise) a non-zero amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.1, said enantiomer E.sub.1 having, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen, under the conditions mentioned below, a retention time t.sub.1 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, said enantiomer E.sub.1 being one of the configurational stereoisomers of one of the two diastereoisomers, said diastereoisomer D.sub.1,4 of flocoumafen, said enantiomer E.sub.1 being different from a configurational stereoisomer of flocoumafen, named enantiomer E.sub.4, said enantiomer E.sub.4 being the other configurational stereoisomer of said diastereoisomer D.sub.1,4 said enantiomer E.sub.4 having, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen, a retention time t.sub.4 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, said composition comprising flocoumafen being free of said enantiomer E.sub.4; the chromatographic analysis being performed at a temperature of 23.5 C. and under the conditions below: on a high-pressure liquid chromatography column of dimensions 1502 mm, and comprising a chiral stationary phase constituted of particles of tris(4-chloro-3-methylphenyl carbamate) cellulose, said particles having a mean size of 3 m and having a mean pore size of 1000 ; using, as liquid mobile phase, a mixture formed from acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with an AB volume ratio of 92/8 and with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL/minute; by injection into the chromatography column of a volume of 1 L of flocoumafen composition at a concentration of 1 g of flocoumafen per millilitre of acetonitrile.

9. Process according to claim 8, wherein the following are chosen in combination: the edible excipient; an amount of said enantiomer E.sub.4 relative to the flocoumafen; a mass proportion of flocoumafen in the rodenticidal bait; and an amount of spread rodenticidal bait; so that target rodent pests consume an amount of flocoumafen that is sufficient to be lethal to said target rodent pests which consume said rodenticidal bait in the course of a single period of 24 consecutive hours.

10. Process according to claim 8, wherein the following are chosen in combination: the edible excipient; an amount of said enantiomer E.sub.4 relative to the flocoumafen; a mass proportion of flocoumafen in the rodenticidal bait; and an amount of spread rodenticidal bait; so that target rodent pests consume an amount of flocoumafen: which is non-lethal for target rodent pests which consume said rodenticidal bait over a single period of 24 consecutive hours; and which is sufficient to be lethal to target rodent pests which consume said rodenticidal bait over several 24-hour periods, said periods being consecutive.

11. Chromatographic process for separating the configurational stereoisomers of flocoumafen, in which: a high-pressure liquid chromatography column of dimensions 1502 mm, and comprising a chiral stationary phase constituted of particles of tris(4-chloro-3-methylphenyl carbamate) cellulose, is chosen, said particles having a mean size of 3 m and having a mean pore size of 1000 ; a mixture formed from acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with an AB volume ratio of 92/8 and with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL/minute, is chosen as liquid mobile phase; separation of the configurational stereoisomers is performed at room temperature, during which: a liquid composition comprising configurational stereoisomers of flocoumafen is introduced into the top of the chromatography column; and then the liquid composition is entrained with the mobile phase in the chromatography column under conditions suitable for separating the configurational stereoisomers of flocoumafen, and a plurality of fractions of the mobile phase are collected, each fraction comprising one of the configurational stereoisomers of flocoumafen separately from the other configurational stereoisomers of flocoumafen; and the liquid mobile phase of each fraction is removed so as to obtain each configurational stereoisomer.

12. Composition according to claim 2, comprising an amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.3, which has, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under the abovementioned conditions, a retention time t.sub.3 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, the amount of said enantiomer E.sub.3 being such that the ratio of this amount to the amount of flocoumafen in the composition is less than 10%.

13. Composition according to claim 1, comprising an amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.1, which has, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under the abovementioned conditions, a retention time t.sub.1 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, the ratio of the amount of said enantiomer E.sub.1 in the composition to the amount of flocoumafen in the composition being between 30% and 100%.

14. Composition according to claim 2, comprising an amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.1, which has, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under the abovementioned conditions, a retention time t.sub.1 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, the ratio of the amount of said enantiomer E.sub.1 in the composition to the amount of flocoumafen in the composition being between 30% and 100%.

15. Composition according to claim 3, comprising an amount of a configurational stereoisomer of flocoumafen, named enantiomer E.sub.1, which has, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under the abovementioned conditions, a retention time t.sub.1 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4, the ratio of the amount of said enantiomer E.sub.1 in the composition to the amount of flocoumafen in the composition being between 30% and 100%.

16. Rodenticidal bait comprising a composition according to claim 2 and at least one excipient that is edible for target rodent pests.

Description

(1) Other aims, characteristics and advantages of the invention will emerge on reading the following description and the examples, which are given for purely non-limiting purposes and which refer to the attached figures, in which:

(2) FIG. 1 is a representative chromatogram of the separation of the enantiomers of flocoumafen on a chiral column;

(3) FIG. 2 is a histogram graph representation of the persistence of the enantiomers of flocoumafen in rat liver.

ANALYSIS OF THE CONFIGURATIONAL STEREOISOMERS OF FLOCOUMAFEN

(4) The configurational stereoisomers of flocoumafen are separated by high-pressure liquid chromatography using a LUX Cellulose-4 chiral column (00F-4490-B0, Phenomenex, Le Pecq, France) as stationary phase and a mixture formed from acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with an A/B volume ratio of 92/8, as mobile phase with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL/minute. 1 L of extract comprising flocoumafen at a concentration of 1 g/mL of acetonitrile is injected. Detection is performed by tandem mass spectrometry (MS/MS) in negative electrospray ionization (ESI) mode. The temperature of the nebulizer gas is 350 C. and its flow rate is 8 L/minute. The pressure of the nebulizer gas is brought to 2700 hPa. In particular, the MRM (Multiple Reaction Monitoring) transitions m/z 541.1.fwdarw.382.1 and m/z 541.1.fwdarw.161, corresponding to the flocoumafen signal, are analysed.

(5) The retention time values for each of the flocoumafen enantiomers are, under the conditions described: t.sub.1=4.5 min for said enantiomer E.sub.1 of said diastereoisomer D.sub.1,4; t.sub.2=6.2 min for said enantiomer E.sub.2 of said diastereoisomer D.sub.2,3; t.sub.3=6.7 min for said enantiomer E.sub.3 of said diastereoisomer D.sub.2,3; and t.sub.4=9.3 min for said enantiomer E.sub.4 of said diastereoisomer D.sub.1,4.

(6) The retention time values t.sub.1, t.sub.2, t.sub.3 and t.sub.4 are liable to vary, especially with the temperature of the chromatography column. However, under these chromatographic conditions, the order of elution of the flocoumafen enantiomers remains unchanged. As a guide, the retention time value (t.sub.1) of enantiomer E.sub.1 of said diastereoisomer D.sub.1,4 may range between 4.4 min and 4.6 min. The retention time value (t.sub.2) of enantiomer E.sub.2 of said diastereoisomer D.sub.2,3 may range between 5.9 min and 6.4 min. The retention time value (t.sub.3) of enantiomer E.sub.3 of said diastereoisomer D.sub.2,3 may range between 6.4 min and 6.9 min. The retention time value (t.sub.4) of said enantiomer E.sub.4 according to the invention may range between 8.9 min and 9.4 min.

(7) Extraction of Flocoumafen from the Liver of Rats Tube-Fed with Flocoumafen

(8) Homogenization of the Liver Sample

(9) About 0.525 g (0.025 g) of rat liver is weighed out accurately and placed in a 50 mL polypropylene tube. 10 mL of acetone are added and the suspension is homogenized using an Ultra-Turrax homogenizer/disperser for a time of about 30 seconds. The homogenizer/disperser shaft is rinsed with hot water and then twice with 20 mL of acetone in a polypropylene tube. The homogenate is centrifuged for 5 minutes at a centrifugation speed of 3000 rpm (revolutions per minute). The supernatant is collected and transferred into a test tube. The sample is subjected to evaporation under a stream of nitrogen (N.sub.2) at a temperature of 40 C. so as to form a dry extract.

(10) Lipid Removal

(11) 1 mL of acetonitrile is added to the tube containing the dry extract so as to dissolve it. The acetonitrile solution is washed twice successively with 1 mL of hexane. The lipid-free extract is dried under a stream of nitrogen (N.sub.2) at a temperature of 40 C. and is then taken up in 0.5 mL of methanol and dissolved by vortex stirring. 0.5 mL of ultra-pure (Milli-Q) water is then added. The sample is vortex-homogenized.

(12) Solid-Phase Extraction (SPE) of Flocoumafen

(13) 1 mL of dichloromethane (CH.sub.2Ct.sub.2), then 1 mL of methanol (CH.sub.3OH), then 1 mL of ultra-pure (Milli-Q) water are passed through an Oasis HLB 1 cc cartridge (WAT094225, Waters). The lipid-free liver extract (1 mL MeOH/Milli-Q H.sub.2O) containing flocoumafen is then loaded onto the top of the cartridge beforehand. The liver extract penetrates through the cartridge by gravity on contact with the solid phase of the cartridge. 1 mL of washing solution formed from methanol (CH.sub.3OH) and ultra-pure water (H.sub.2O) in a 90/10 volume proportion is loaded onto the top of the cartridge. The cartridge is dried by suction under vacuum connected to the bottom of the cartridge. 1 mL of eluting solution formed from dichloromethane (CH.sub.2Cl.sub.2) and methanol (CH.sub.3OH) in a 90/10 volume proportion is then loaded onto the top of the cartridge and an eluate comprising flocoumafen is collected at the bottom of the cartridge. The solvent of the eluate is evaporated off under a stream of nitrogen (N.sub.2) at a temperature of 40 C. The sample is taken up in 0.5 mL of acetonitrile (NCCH.sub.3) and the acetonitrile solution containing flocoumafen is filtered through a 0.2 m porosity filter.

(14) Hepatic Persistence of the Configurational Stereoisomers (E.sub.1, E.sub.2, E.sub.3 and E.sub.4) of Flocoumafen in Rats

(15) A solution comprising flocoumafen in a mixture of vegetable oil and 5% of DMSO so that the amount of flocoumafen ingested by each rat is about 2.3 mg per kilogram of rat is administered orally (per os) to 8-week-old male and female laboratory rats (Sprague-Dawley rats, Charles River, Saint-Germain-sur-l'Arbresle, France) weighing between 180 and 200 g. The tube-fed rats are treated daily by subcutaneous administration of a dose of vitamin K1 at a rate of 1 U per rat so as to keep the rats alive throughout the experiment.

(16) The ratio of the sum of the amounts of said enantiomer E.sub.2 and of said enantiomer E.sub.3 (diastereoisomer D.sub.2,3) to the amount of flocoumafen in the tube-feeding solution is 59% and the ratio of the sum of the amounts of said enantiomer E.sub.1 and of said enantiomer E.sub.4 (diastereoisomer D.sub.1,4) to the amount of flocoumafen in the tube-feeding solution is 41%.

(17) At 1 day (D+1), 3 days (D+3) and 7 days (D+7) after tube-feeding, three male rats and three female rats anaesthetized beforehand with isoflurane are euthanized, the liver of the sacrificed rats is removed and the amounts of each of the configurational stereoisomers of flocoumafen present in the liver of the tube-fed male and female rats are then extracted from the liver and assayed. The results obtained on the male rats are given in table 1 below and the results obtained on the female rats are given in table 2 below in which the values of the concentration of each of the configurational stereoisomers of flocoumafen in the liver are the mean of the values measured on three rats expressed in nanograms (ng) per gram of liver.

(18) TABLE-US-00001 TABLE 1 (male rats) Hepatic concentration, ng/g D + 1 D + 3 D + 7 Enantiomer E.sub.1 196 47 0 Enantiomer E.sub.2 3904 1962 319 Enantiomer E.sub.3 3301 1862 647 Enantiomer E.sub.4 8143 9906 2623

(19) TABLE-US-00002 TABLE 2 (female rats) Hepatic concentration, ng/g D + 1 D + 3 D + 7 Enantiomer E.sub.1 4585 2692 1196 Enantiomer E.sub.2 5396 2522 469 Enantiomer E.sub.3 5478 2734 1228 Enantiomer E.sub.4 10342 7765 5054

(20) FIG. 2 represents the change in the percentage of the mass amount of each enantiomer (E.sub.1 and E.sub.4) of diastereoisomer D.sub.1,4 and of each enantiomer (E.sub.2 and E.sub.3) of diastereoisomer D.sub.2,3 relative to the amount of flocoumafen retained in the liver of the tube-fed rats (male and female) sacrificed on D+1, D+3 and D+7. The percentage of said enantiomer E.sub.1 is represented by oblique-hatched columns, the percentage of said enantiomer E.sub.4 is represented by horizontally-hatched columns, the percentage of said enantiomer E.sub.2 is represented by black columns and the percentage of said enantiomer E.sub.3 is represented by white columns. The respective proportions of each of the enantiomers (E.sub.1 and E.sub.4) of diastereoisomer D.sub.1,4 and of the enantiomers (E.sub.2 and E.sub.3) of diastereoisomer D.sub.2,3 in the flocoumafen of the tube-feeding composition are represented in column X FIG. 2 and are 20.5% for enantiomer E.sub.1 of said diastereoisomer D.sub.1,4 and 20.5% for enantiomer E.sub.4 of said diastereoisomer D.sub.1,4 and 29.5% for said enantiomer E.sub.2 and 29.5% for said enantiomer E.sub.3. Said enantiomer E.sub.4 is the flocoumafen enantiomer that is the most persistent in rat liver and the most toxic to the environment. The invention is thus directed towards a composition, a rodenticidal bait and a process for controlling target rodent pests comprising a small content of enantiomer E.sub.4 different from the content of enantiomer E.sub.1.

(21) It goes without saying that the invention may be the subject of numerous implementation variants and applications. In particular, a composition, a rodenticidal bait and a process for controlling target rodent pests are subject to an infinite number of variants both in the formulation of the rodenticidal bait and in the embodiments of the process.