Rodenticide bait comprising difenacoum and method for controlling target rodent pests

Abstract

Disclosed is a rodenticide bait including: difenacoum in the form of trans-difenacoum of formula 3-(biphenyl-4-yl)-1 -(4-hydroxycoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene, in wherein carbons 1 and 3 of group 1,2,3,4-tetrahydronaphthalene of the trans-difenacoum have the same absolute configuration; and an edible excipient for target rodent pests. The bait includes trans-difenacoum at a concentration above a minimum trans-difenacoum concentration lethal to female adults of the target rodents, but below a minimum trans-difenacoum concentration lethal to adult males of the target rodents, the minimum concentration lethal to female adults being less than the minimum concentration lethal to male adults. Also disclosed is a method for selectively controlling a population of target rodent pests.

Claims

1. Rodenticidal bait comprising: difenacoum in a majority in the form of trans-difenacoum having the formula 3-(biphenyl-4-yl)-1-(4-hydroxycoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene, in which carbon atoms 1 and 3 of the 1,2,3,4-tetrahydronaphthalene grouping of trans-difenacoum have the same absolute configuration, and; an excipient which is edible for target rodent pests; wherein a proportion of trans-difenacoum in the bait is greater than a minimum proportion of trans-difenacoum which is lethal to adult females of the target rodents and less than a minimum proportion of trans-difenacoum which is lethal to adult males of the target rodents, said minimum proportion which is lethal to adult females being less than the minimum proportion which is lethal to adult males.

2. Bait according to claim 1, wherein the excipient which is edible comprises at least one food selected from the group consisting of cereal grains, ground cereal grains, cereal grain flours, cereal grain flakes, cereal bran, non-cereal grains, ground non-cereal grains, non-cereal grain flours, non-cereal grain flakes, and non-cereal plant bran.

3. Bait according to claim 1, having a proportion by mass of trans-difenacoum of between 1 ppm and 30 ppm.

4. Method of selective combating of a population of target rodent pests by scattering a bait liable to be ingested by the target rodent pests, said bait comprising: difenacoum in a majority in the form of trans-difenacoum having the formula 3-(biphenyl-4-yl)-1-(4-hydroxycoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene, in which carbon atoms 1 and 3 of the 1,2,3,4-tetrahydronaphthalene grouping of trans-difenacoum have the same absolute configuration, and an excipient which is edible for target rodent pests; wherein a proportion of trans-difenacoum in the bait is greater than a minimum proportion which is lethal to adult females of the target rodents and less than a minimum proportion which is lethal to adult males of the target rodents, said minimum proportion which is lethal to adult females being less than the minimum proportion which is lethal to adult males, and wherein these baits are scattered in an amount sufficient to be lethal to adult females of the target rodents.

5. Method according to claim 4, wherein the proportion of trans-difenacoum in the bait and the amount of bait scattered are adjusted to achieve: in the adult females of the target rodent pests, an amount of trans-difenacoum which is lethal to said adult females, and in the adult males of the target rodent pests, an amount of trans-difenacoum which is not lethal to said adult males.

6. Method according to claim 4, wherein the proportion of trans-difenacoum in the bait is chosen in combination with the amount of bait scattered such that adult females of the target rodents eat during a single period of 24 consecutive hours an amount of bait sufficient to be lethal to said adult females of the target rodents which eat said bait.

7. Method according to claim 4, wherein the proportion of trans-difenacoum in the bait is chosen in combination with the amount of bait scattered such that the adult females of target rodent pests eat an amount of difenacoum which is: non-lethal to the adult females of target rodent pests which eat said bait during a period of 24 consecutive hours, and sufficient to be lethal to the adult females of target rodent pests which eat said bait during several periods of 24 consecutive hours, said periods being successive.

8. Method according to claim 7, wherein the amount of trans-difenacoum which is lethal to the adult female(s) is achieved after ingestion of a plurality of daily doses of said bait by the adult female(s).

9. Method according to claim 4, wherein the proportion of trans-difenacoum in the bait and the amount of bait scattered are chosen in order to be able to achieve in the liver of the adult females, on the day following the third day of a period of three consecutive days, each day of said period comprising at least one ingestion of bait, an amount of trans-difenacoum of less than or equal to 10 g of trans-difenacoum per gram of liver of said adult females.

10. Method according to claim 4, wherein the bait is chosen in order to allow preferential poisoning of the adult females of target rodent pests.

11. Bait according to 2, having a proportion by mass of trans-difenacoum of between 1 ppm and 30 ppm.

12. Method according to claim 5, wherein the proportion of trans-difenacoum in the bait is chosen in combination with the amount of bait scattered such that adult females of the target rodents eat during a single period of 24 consecutive hours an amount of bait sufficient to be lethal to said adult females of the target rodents which eat said bait.

13. Method according to claim 5, wherein the proportion of trans-difenacoum in the bait is chosen in combination with the amount of bait scattered such that the adult females of target rodent pests eat an amount of difenacoum which is: non-lethal to the adult females of target rodent pests which eat said bait during a period of 24 consecutive hours, and sufficient to be lethal to the adult females of target rodent pests which eat said bait during several periods of 24 consecutive hours, said periods being successive.

14. Method according to claim 5, wherein the proportion of trans-difenacoum in the bait and the amount of bait scattered are chosen in order to be able to achieve in the liver of the adult females, on the day following the third day of a period of three consecutive days, each day of said period comprising at least one ingestion of bait, an amount of trans-difenacoum of less than or equal to 10 g of trans-difenacoum per gram of liver of said adult females.

15. Method according to claim 6, wherein the proportion of trans-difenacoum in the bait and the amount of bait scattered are chosen in order to be able to achieve in the liver of the adult females, on the day following the third day of a period of three consecutive days, each day of said period comprising at least one ingestion of bait, an amount of trans-difenacoum of less than or equal to 10 g of trans-difenacoum per gram of liver of said adult females.

16. Method according to claim 7, wherein the proportion of trans-difenacoum in the bait and the amount of bait scattered are chosen in order to be able to achieve in the liver of the adult females, on the day following the third day of a period of three consecutive days, each day of said period comprising at least one ingestion of bait, an amount of trans-difenacoum of less than or equal to 10 g of trans-difenacoum per gram of liver of said adult females.

17. Method according to claim 8, wherein the proportion of trans-difenacoum in the bait and the amount of bait scattered are chosen in order to be able to achieve in the liver of the adult females, on the day following the third day of a period of three consecutive days, each day of said period comprising at least one ingestion of bait, an amount of trans-difenacoum of less than or equal to 10 g of trans-difenacoum per gram of liver of said adult females.

Description

(1) Other objects, characteristics and advantages of the invention will emerge from reading the following description and the examples given purely as non-limiting and which refer to the attached figures, in which:

(2) FIG. 1 is a proton NMR spectrum of trans-difenacoum;

(3) FIG. 2 is a proton NMR spectrum of cis-difenacoum;

(4) FIG. 3 is a comparative graphical representation of the hepatic persistence of trans-difenacoum and of cis-difenacoum in the rat;

(5) FIG. 4 is a graphical representation of the hepatic persistence (in g/g of liver) at D.sub.4 and the Quick time (QT) of male rats which have received treatment with three doses (D.sub.1, D.sub.2 and D.sub.3) of a solution comprising trans-difenacoum administered in an amount of 0.6; 1.2 or 2.6 mg of difenacoum per kilogram of rat;

(6) FIG. 5 is a graphical representation of the hepatic persistence (in g/g of liver) at D.sub.4 and the Quick time (QT) of female rats which have received treatment with three doses (D.sub.1, D.sub.2 and D.sub.3) of a solution comprising trans-difenacoum administered in an amount of 0.3; 0.6; 0.75; 1.2 or 2.6 mg of difenacoum per kilogram of rat.

(7) A rodenticidal bait according to the invention comprises trans-difenacoum as the rodenticidal agent and at least one attractant substance for target rodent pests, said attractant substance being capable of stimulating the appetite of species of target rodent pests and the ingestion of said bait by the target rodent pests.

(8) trans-Difenacoum is obtained by any method allowing the separation of the cis/trans configurational stereoisomers of difenacoum. For example, the document EP 0 175 466 discloses a method for the separation of the trans and cis configurational stereoisomers of difenacoum by selective precipitation of the cis configurational stereoisomer from a solution of the configurational stereoisomers in an alcohol such as ethanol.

(9) It is also possible to separate the trans-difenacoum and cis-difenacoum configurational stereoisomers by chromatography, for example by high performance liquid chromatography (HPLC), and to form a mixture enriched in trans-difenacoum or a composition of trans-difenacoum essentially free from cis-difenacoum. Such an enrichment is carried out by HPLC, for example on a C8 (250 mm10 mm) column kept at the temperature of 30 C. The mobile phase is formed from 80% acetonitrile and 20% acid water. The flow rate of the mobile phase is kept at 2 ml/min. The trans-difenacoum and the cis-difenacoum are detected by spectrophotometric analysis at the wavelength of 258 nm.

(10) In particular, such a separation of the trans-difenacoum and cis-difenacoum configurational stereoisomers is described, for example, in the publication Kelly M. J. et al., (1993), Journal of Chromatography, 620, 105-112. Simple and rapid method for the determination of the diastereoisomers of difenacoum in blood and liver using high performance liquid chromatography with fluorescence detection. This document also reports that the cis and trans configurational stereoisomers of difenacoum differ in proton NMR (Brker AC 300 spectrometer at 300.13 MHz in CDCl.sub.3), the signal corresponding to the proton of carbon atom 1 of the 1,2,3,4-tetrahydronaphathalene grouping of trans-difenacoum being a triplet centred at 4.76 ppm and having a coupling constant of 4.20 Hz and the signal of the proton of carbon atom 1 of the 1,2,3,4-tetrahydronaphathalene grouping of cis-difenacoum being a quartet centred at 4.9 ppm and having coupling constants of 11.9 and 6 Hz with the protons of carbon atom 2 of the 1,2,3,4-tetrahydronaphathalene grouping.

(11) It is also possible to carry out an enrichment of trans-difenacoum by HPLC chromatography on an INERTSIL ODS2 reverse phase column of 150 mm length and 4.6 mm internal diameter. The reverse stationary phase has a particle size of 5 m, a porosity of 80 and a specific surface area of 500 m.sup.2/g. The mobile phase is made up of a mixture of acetonitrile (62%), isopropanol (3%) and ammonium acetate (35%) adjusted to pH 4 with acetic acid. The flow rate of the mobile phase is 1.5 ml/min and the detection is carried out by spectrophotometry at 260 nm. A trans configurational stereoisomer of which the retention time is of the order of 5.74 min and another cis configurational stereoisomer of which the retention time is of the order of 4.87 min are detected and collected.

(12) The separation of the trans and cis configurational stereoisomers of difenacoum can also be carried out by ultra-performance liquid chromatography (UPLC, Waters) on an Acquity Waters chain comprising an Acquity UPLC BEH C18 column of 1.7 m particle size and dimensions of 2.150 mm at 35 C. The mobile phase is formed by a gradient of an aqueous solution of trifluoroacetic acid (TFA, 0.1%) and a solution of TFA, 0.1% in acetonitrile. The flow rate of the mobile phase is 0.6 ml/min. The retention time of the configurational stereoisomer retained the most (trans) is 8.2 min and that of the configurational stereoisomer retained the least (cis) is of the order of 7.3 min.

(13) The separation of the trans and cis configurational stereoisomers of difenacoum can also be carried out by high pressure liquid chromatography (HPLC) on an XBridge C18 column (dimensions of 4.6150 mm and particle size of 5 m) and with a mobile phase formed by 45% of an aqueous 0.1% solution of TFA and 55% of a solution of TFA at 0.1% in acetonitrile. The flow rate of the mobile phase is 1.2 ml/min. The retention time of the configurational stereoisomer retained the most (trans) is 23.7 min and that of the configurational stereoisomer retained the least (cis) is of the order of 21.0 min.

(14) The proton NMR spectrum of the trans configurational stereoisomer of difenacoum is shown in FIG. 1 and the proton NMR spectrum of the cis configurational stereoisomer of difenacoum is shown in FIG. 2. The proton NMR spectrum of trans-difenacoum has a triplet (4.5399 ppm, 4.5644 ppm and 4.5876 ppm) characteristic of the proton carried by carbon atom 1 of the 1,2,3,4-tetrahydronaphathalene grouping. By comparison, the proton NMR spectrum of cis-difenacoum has a quartet (4.8180 ppm, 4.8374 ppm, 4.8550 ppm and 4.8721 ppm) characteristic of the proton carried by carbon atom 1 of the 1,2,3,4-tetrahydronaphathalene grouping.

(15) Hepatic Persistence Compared for the Trans and Cis Configurational Stereoisomers of Difenacoum in the Rat

(16) On D0 per os tube feeding is performed on male laboratory rats (Sprague Dawley rats, Charles River, Saint germain sur 1 Arbresle, France) aged 8 weeks and with a body mass of the order of 200 g with a mixture comprising 56% of trans-difenacoum and 44% of cis-difenacoum in an amount of 5.2 mg of difenacoum per kilogram of rat. The rats are kept alive by daily subcutaneous injection of a solution of vitamin K in an amount of 1 U per rat. On D+1, D+3, D+5, D+7, D+10, D+14 and D+21, 4 rats are anaesthetised with isoflurane and then sacrificed, and the livers are removed and frozen until analysed. The mean hepatic contents of trans-difenacoum and cis-difenacoum are analysed by HPLC and are given in Table 1 below.

(17) TABLE-US-00001 TABLE 1 Hepatic content, g/g D + 1 D + 3 D + 5 D + 10 D + 21 trans 4.22 1.05 0.25 0.1 0.17 cis 16.82 8.28 4.86 1.85 1.41

(18) From D+1 the mean content of trans-difenacoum in the liver of rats is lower than the mean content of cis-difenacoum in the livers of rats tube-fed with essentially the same amount of each configurational stereoisomer of difenacoum. trans-Difenacoum has a hepatic persistence significantly lower than that of cis-difenacoum.

(19) An amount of trans-difenacoum is dispersed in an amount of an attractant and nutritive substance for target rodent pests by any method known per se to the person skilled in the art. The amounts of trans-difenacoum and attractant substance are chosen such as to form a rodenticidal bait according to the invention comprising trans-difenacoum in a proportion greater than the minimum proportion which is lethal in less than 10 days to the adult females of the target rodent pests and non-lethal to the adult males of the target rodent pests.

(20) Such a proportion of trans-difenacoum in the rodenticidal bait is adjusted so that female target rodent pests eating the rodenticidal bait, the attractant substance and the trans-difenacoum, ingest an amount of trans-difenacoum which is sufficient to obtain the rodenticidal effect and to control the population of target rodent pests.

(21) 1) Comparison of the Rodenticidal Efficacy of Trans-Difenacoum and Cis-Difenacoum in the Male Rat and the Female Rat.

(22) The comparative study of the rodenticidal efficacy of purified trans-difenacoum and purified cis-difenacoum is carried out by administration by means of a stomach tube (per os): of a solution of cis-difenacoum to a first group of four Sprague Dawley male rats weighing between 180 and 220 g, and of a solution of trans-difenacoum to a second group of four Sprague Dawley male rats weighing between 180 and 220 g.

(23) These solutions of cis-difenacoum and trans-difenacoum are obtained by weighing cis-difenacoum or trans-difenacoum and dilution in a mixture formed from 95% vegetable oil and 5% DMSO.

(24) The doses of cis-difenacoum and trans-difenacoum used are 2.6 mg of cis-difenacoum or trans-difenacoum per kilogram of rat. The dose of 2.6 mg of cis-difenacoum and trans-difenacoum per kilogram of rat corresponds to 4 times the median lethal dose (LD.sub.50) of difenacoum for rats. The dose of 5.2 mg of cis-difenacoum or trans-difenacoum per kilogram of rat corresponds to 8 times the median lethal dose (LD.sub.50) of difenacoum for rats.

(25) 3 ml blood samples are taken from the rats anaesthetised with isoflurane in sample tubes containing citrate (3.2%). The tubes are centrifuged at 2,000 g for 10 minutes and the plasma fraction is collected.

(26) The coagulation time (Quick time) of the plasma is analysed by means of the Neoplastine CI Determination of Prothrombin Time kit (Diagnostica Stago, Asnire, France) by measurement on a Thrombotimer option 2 plus (Behnk Electronik, Norderstedt, Germany).

(27) The normal value for the coagulation time of untreated rats is 10 to 20 seconds.

(28) The administration (D.sub.0) per os of cis-difenacoum in an amount of 2.6 mg of cis-difenacoum per kilogram of rat or of trans-difenacoum in this same dosage of 2.6 mg/kg of rat causes a significant increase in the coagulation time of the rats at 1 day after the administration (D.sub.+1).

(29) The coagulation time (D.sub.+1) of the blood of rats treated with a dosage of 2.6 mg of cis-difenacoum or trans-difenacoum per kilogram of rat is 62 seconds. In contrast, the administration per os of trans-difenacoum in this same dosage (2.6 mg/kg) or in double the dosage (5.2 mg/kg) certainly causes an increase in the coagulation time (62 s and 84 s) on D.sub.+1, but leads to a value of the Quick time on D.sub.+3 which is low (10 to 20 s) and of the order of size of the Quick time of an untreated control. No indication was therefore given to the inventors to choose a rodenticidal bait comprising trans-difenacoum in a majority.

(30) In addition, the value of the inhibition constant (Ki.sub.cis=60 nM) of the recombinant enzyme rVKORC1 by cis-difenacoum is similar to the value of the inhibition constant of rVKORC1 by trans-difenacoum (Ki.sub.trans=30 nM).

(31) 2) Hepatic Persistence of Cis-Difenacoum and Trans-Difenacoum in the Male Rat.

(32) On D.sub.0 a control composition of the prior art comprising a molar proportion of cis-difenacoum of 56% and a molar proportion of trans-difenacoum of 44% is administered per os to seven groups of four male laboratory rats (Sprague Dawley rats, Charles River, Saint germain sur l'Arbresle, France) weighing between 180 and 220 g in an amount of 5.2 mg of difenacoum per kilogram of rat. On D.sub.+1, D.sub.+3, D.sub.+5, D.sub.+7, D.sub.+10, D.sub.+14 and D.sub.+21, 4 rats are sacrificed. The livers are removed and weighed. A liquid/solid extraction of difenacoum with acetone is performed. The difenacoum is analysed by HPLC on a C8 (250 mm10 mm) column kept at 30 C. with a mobile phase formed from 80% acetonitrile and 20% acid water. The flow rate of the mobile phase is kept at 2 ml/min and the trans-difenacoum and cis-difenacoum are detected by spectrophotometric analysis at the wavelength of 258 nm. The trans-difenacoum and cis-difenacoum are quantified by calibration with known amounts of trans-difenacoum and cis-difenacoum. The results are given in FIG. 3, in which the hepatic persistence of trans-difenacoum is represented by solid squares (.square-solid.) and the hepatic persistence of cis-difenacoum is represented by open diamonds (). Whereas the molar proportion of trans-difenacoum administered to the rats is 46%, the hepatic concentration (expressed in g of difenacoum per g of liver) of trans-difenacoum at D.sub.+1 is only of the order of 1 g/g, trans-difenacoum being almost undetectable from D.sub.+3, only the cis-difenacoum remaining (4 g/g).

(33) 3) Hepatic Persistence of Trans-Difenacoum and Cis-Difenacoum in the Male Rat and in the Female Rat.

(34) A study comparable to that described under 2), in which a separation and an assay of trans-difenacoum and cis-difenacoum are carried out on D.sub.+1, D.sub.+3, D.sub.+5, D.sub.+7, D.sub.+10, D.sub.+14 and D.sub.+21, is conducted on treated male rats and female rats. On D.sub.+1 the concentration of cis-difenacoum in the liver of the female rats (9.6 g/g of liver) is essentially equivalent to the concentration of cis-difenacoum in the liver of the male rats (7.3 g/g of liver). The concentration on D.sub.+1 of trans-difenacoum in the liver of the female rats (3.3 g/g of liver) is greater than the concentration of trans-difenacoum in the liver of the male rats (0.9 g/g of liver). In addition, on D.sub.+3 the value of the concentration of trans-difenacoum in the liver is greater in the female rats in comparison with the male rats. The kinetics of the hepatic elimination of trans-difenacoum are slower in the female rats than in the male rats, in particular during the first 3 days. For the same dosage, the hepatic concentrations of trans-difenacoum are thus higher in the female at least up to three days after treatment (D.sub.0).

(35) 4) Administration of Trans-Difenacoum to Male Rats, Multi-Dose Treatment (Multi-Feeding).

(36) Counting from D.sub.0 and for three days (D.sub.0, D.sub.+1 and D.sub.+2), trans-difenacoum in an amount of 0.6 mg/kg, 1.2 mg/kg or 2.6 mg/kg (mg of trans-difenacoum per kilogram of male rat) is administered daily to Sprague Dawley male rats weighing between 180 and 220 g. On D.sub.+3, the coagulation time of these male rats is measured and the concentration of trans-difenacoum in the liver of these male rats (expressed in g of trans-difenacoum per g of liver) is analysed. The results are given in FIG. 4. The administration for three days (D.sub.0, D.sub.+1 and D.sub.+2) in an amount of one dose of trans-difenacoum per day to male rats gives rise to a massive increase in the Quick time of these rats on D.sub.+3 and a moderate value of the hepatic persistence of the order of 2 g/g of liver.

(37) With a dosage of 1.2 mg/kg or higher (corresponding to a bait dosed with between 12 ppm and 25 ppm of trans-difenacoum), the increase in the Quick time is such that the blood of the male rat is uncoagulable. The probability that this male rat dies of haemorrhage is thus a maximum.

(38) 5) Administration of Trans-Difenacoum to Female Rats. Multi-Dose Treatment (Multi-Feeding).

(39) Counting from D.sub.0 and for three days (D.sub.0, D.sub.+1 and D.sub.+2), trans-difenacoum in an amount of 0.3 mg/kg; 0.6 mg/kg; 0.75 mg/kg; 1.2 mg/kg or 2.6 mg/kg (mg of trans-difenacoum per kilogram of female rat) is administered to female Sprague Dawley rats in an amount of one dose per day. On D.sub.+4, the coagulation time of these female rats is measured and the concentration of trans-difenacoum in the liver (expressed in g of difenacoum per g of liver) of these female rats is analysed. The results are given in FIG. 5. The administration for three days (D.sub.0, D.sub.+1 and D.sub.+2) in an amount of a dose of trans-difenacoum greater than 0.3 mg/kg/day to female rats achieves at D.sub.+4 a high value of the coagulation time (greater than or equal to 500 seconds) in these female rats and a moderate hepatic persistence of less than 10 g of trans-difenacoum per gram of liver for a dose of less than 2.6 mg/kg.

(40) With a dosage of 0.6 mg/kg or higher (corresponding to a rodenticidal bait dosed with between 6 ppm and 25 ppm of trans-difenacoum), the increase in the coagulation time is such that the blood of the female rats is uncoagulable. The probability that these female rats die of haemorrhage is thus a maximum.

(41) A rodenticidal bait comprising trans-difenacoum in a majority and a method of combating target rodent pests using such a rodenticidal bait according to the invention allows an anticoagulant effect to be obtained on repeated administration (multi-dose) of trans-difenacoum and very low hepatic concentrations in the male rats with a dosage of 3 times 2.6 mg/kg and in the female rats with a dosage of less than 2.6 mg/kg.

(42) The hepatic concentration of trans-difenacoum is of the order of 2 g/g of liver of the male rat the day following the last dose of rodenticidal bait. Considering that the mass of a rat liver is of the order of 10 g, the global amount of trans-difenacoum is 20 g per liver, and considering that the liver contains half the trans-difenacoum ingested by the rodent, a rat corpse would contain about 40 g of trans-difenacoum. Consequently, the corpses of rats killed by ingestion of a bait according to the invention present a reduced ecotoxicological risk with respect to corpses of rats killed by ingestion of a bait of the prior art.

(43) By way of example, the hepatic concentration of trans-difenacoum of female rats treated with a bait allowing the administration of 2.6 mg of trans-difenacoum per day and per kilogram of rat for 3 days is of the order of 12 g/g of liver. Since predators of rodents rapidly eliminate trans-difenacoum, a corpse of a female target rodent pest containing such a dose of trans-difenacoum is much less dangerous to the environment than a corpse of a female target rodent pest containing the same amount of the cis-difenacoum configurational stereoisomer.

(44) Thus, the use of trans-difenacoum in a bait in which trans-difenacoum is in a majority is less dangerous to species exposed to the risk of primary intoxication (ingestion of the bait according to the invention by a non-target animalby a dog, for example). Eating a bait according to the invention containing trans-difenacoum in a majority is liable to present problems to a non-target animal only if said non-target animal can have access to significant amounts of said rodenticidal bait repeatedly over time. The risk is thus reduced considerably with respect to the use of a commercial mixture which is liable to cause a fatal intoxication in the dog in a single dose.

(45) 6) Treatment of a Population of Rats with a Bait According to the Invention Comprising 50 ppm of Trans-Difenacoum.

(46) Rodenticidal baits are prepared by dispersing an amount of difenacoum in a paste based on flour and vegetable fat such that the bait contains 50 mg of difenacoum per kilogram of bait (50 ppm of difenacoum).

(47) The following are prepared: a rodenticidal bait according to the invention comprising trans-difenacoum in a majority (91% of trans-difenacoum and 9% of cis-difenacoum), and by way of comparison baits comprising: 4% of trans-difenacoum and 96% of cis-difenacoum, and 38% of trans-difenacoum and 62% of cis-difenacoum.

(48) Rodents (10 Sprague Dawley (SD) rats, 5 males and 5 females) are placed in individual cages. During 4 days (D1, D2, D3 and D4) each rat is supplied with an amount of bait sufficient to satisfy its appetite. At the end of these 4 days food free from bait is provided. The daily consumption of each bait is measured by weighing. On the death of the animal the liver of the dead animal is removed, frozen and stored until the hepatic content of configurational stereoisomers of difenacoum is analysed.

(49) The mean daily consumption of bait by the rats and the time at which the death of the animals occurs and the mortality are given in Table 2 below.

(50) TABLE-US-00002 TABLE 2 trans-Difenacoum/ Mean mass of bait Time of Mortality, cis-Difenacoum consumed daily, g/day death % 91/9 12.9 D4 to D8 90 38/62 12.5 D4 to D9 90 4/96 11.8 D5 to D9 100

(51) The bait according to the invention (91/9) is eaten by the rats with the same mean daily amount as the baits (2/98 or 18/82) not enriched in trans-difenacoum. The time at which death occurs in the animals having eaten the bait according to the invention is shorter (D4-D8) than the time at which death occurs in the animals having eaten baits 38/62 (D4 to D9) and 4/96 (D5 to D9).

(52) The results of the hepatic assays of trans-difenacoum and cis-difenacoum are presented in Table 3 below, in which trans-difenacoum/cis-difenacoum represents the amount (molar or by mass) of trans-difenacoum relative to the amount (molar or by mass) of cis-difenacoum in the bait made available to the rats and the term Residue represents the percentage of difenacoum (trans-difenacoum and cis-difenacoum) detected in the liver of the rats relative to the total mean amount of difenacoum ingested by each rat.

(53) TABLE-US-00003 TABLE 3 trans-Difenacoum/ Hepatic content, g/g Residue, cis-Difenacoum trans-Difenacoum cis-Difenacoum % 91/9 0.76 0.63 0.4 38/62 0.15 1.54 0.59 4/96 0.01 2.16 0.7

(54) The mean content of difenacoum in the liver of the male rats is 8.4 s (4.3 s of trans-difenacoum and 4.1 g of cis-difenacoum), 13.5 s (1.4 s of trans-difenacoum and 12.1 s of cis-difenacoum) and 21.9 s (0.2 s of trans-difenacoum and 21.7 s of cis-difenacoum) per male rat treated, respectively, with the baits (91/9), (38/62) and (4/96). The residues of difenacoum in the liver of the male rats are minimised with the use of the bait according to the invention.

(55) The mean content of difenacoum in the liver of the female rats is 14.9 s (8.5 s of trans-difenacoum and 6.4 g of cis-difenacoum), 16.4 s (1.1 g of trans-difenacoum and 15.2 g of cis-difenacoum) and 10.8 g (10.7 g of cis-difenacoum) per female rat treated with, respectively, the baits (91/9), (38/62) and (4/96). The residues of difenacoum in the liver of the female rats are minimised with the use of the bait according to the invention. The residues of difenacoum in the liver of the male rats are also minimised with respect to the residues of difenacoum in the liver of the female rats with the use of the bait (91/9) according to the invention.

(56) A bait (91/9) comprising trans-difenacoum in a majority thus allows limiting of the residues of difenacoum in the liver of rodents and their secondary toxicity to predators of rodents or carrion eaters of corpses of rodents.

(57) 7) Treatment of a Population of Rats with a Bait According to the Invention Comprising 15 ppm of Trans-Difenacoum.

(58) Rodenticidal baits are prepared by dispersing an amount of difenacoum in a paste based on flour and vegetable fat such that the bait contains 15 mg of difenacoum per kilogram of bait (15 ppm of difenacoum).

(59) The following are prepared: a rodenticidal bait according to the invention comprising trans-difenacoum in a majority (essentially 100% of trans-difenacoum), and by way of comparison a bait comprising 100% of cis-difenacoum.

(60) Rodents (10 Sprague Dawley (SD) rats, 5 males and 5 females) are placed in individual cages and treated as described under 6) above. The mean daily consumption of bait by the rats and the time at which the death of the animals occurs and the mortality are given in Table 4 below.

(61) TABLE-US-00004 TABLE 4 trans-Difenacoum/ Mean mass of bait Time of Mortality, cis-Difenacoum consumed daily, g/day death % 100/0 14.8 D5 to D8 70% 0/100 13.8 D4 to D9 100%

(62) The bait comprising 15 ppm of difenacoum according to the invention (essentially of the order of 100% trans-difenacoum in the difenacoum) is eaten by the rats with the same mean daily amount as the bait (100% cis-difenacoum). The time at which death occurs in the animals having eaten the bait according to the invention is comparable (D5-D8) to the time at which death occurs in the animals having eaten the bait having a low dose of trans-difenacoum (D4 to D9).

(63) A bait according to the invention comprising trans-difenacoum in a majority thus allows limiting of the residues of difenacoum in the liver of rodents and their secondary toxicity to predators of rodents or carrion eaters of corpses of rodents while preserving a rodenticidal efficacy of the order of 70% with a bait having a low dose of 15 ppm.

(64) It goes without saying that the invention may be subject to numerous embodiments and applications. In particular, a rodenticidal bait and a method of combating at least one species of target rodent pests are subject to an infinite number of variants both in the formulation of the bait and in the modes of implementation of the method.