RODENTICIDAL BAIT AND METHOD OF CONTROLLING TARGET HARMFUL RODENTS

Abstract

The invention relates to a rodenticidal bait by ingestion for at least one target harmful rodent ingesting this bait for an ingestion period, called consumption time, imposed owing to said ingestion; the rodenticidal bait comprising: a composition, called composition of AVK(s), of at least one compound that inhibits vitamin K recycling; a composition, called vitamin D composition, of at least one hypercalcaemia-causing vitamin D, and at least one excipient that is edible for target harmful rodents;
characterized in that: said composition of AVK(s) is present in the rodenticidal bait in a proportion by weight, called proportion of AVK(s) by weight, that is not zero and is not lethal on its own for any target harmful rodent, and in that said vitamin D composition is present in the rodenticidal bait in a proportion by weight, called the proportion of vitamin D by weight: sufficient for said vitamin D composition to be lethal on its own for at least one target harmful rodent, and below a minimum proportion by weight sufficient for said vitamin D composition to be lethal on its own for any target harmful rodent. The invention also relates to the uses of a rodenticidal bait according to the invention and a method of controlling target harmful rodents.

Claims

1. A rodenticidial bait by ingestion for at least one target harmful rodent ingesting this bait for an ingestion period, called consumption time, imposed owing to said ingestion; the rodenticidal bait comprising: a composition, called composition of AVK(s), of at least one compound that inhibits vitamin K recycling; a composition, called vitamin D composition, of at least one hypercalcaemia-causing vitamin D, and at least one excipient that is edible for target harmful rodents; wherein: said composition of AVK(s) is present in the rodenticidal bait in a proportion by weight, called proportion of AVK(s) by weight, that is not zero and is not lethal on its own for any target harmful rodent ingesting this bait for said consumption time, and in that said vitamin D composition is present in the rodenticidal bait in a proportion by weight, called the proportion of vitamin D by weight: sufficient for said vitamin D composition to be lethal on its own for at least one target harmful rodent, and below a minimum proportion by weight sufficient for said vitamin D composition to be lethal on its own for any target harmful rodent.

2. The bait according to claim 1, wherein said composition of AVK comprises at least one compound selected from the group comprising 4-hydroxycoumarins substituted in position 3, 4-hydroxythiocoumarins substituted in position 3 and indan-1,3-diones substituted in position 2.

3. The bait according to claim 1, wherein said composition of AVK(s) comprises at least one 4-hydroxycoumarin substituted in position 3 selected from the group comprising coumachlor, warfarin, coumatetralyl, dicoumarol, acenocoumarol, phenprocoumon, 3-(1-(4′-fluorobiphenyl yl)-ethyl)-4-hydroxy-2H-1-benzopyran-2-one, of formula (Chem.4) hereunder, ##STR00016## compounds of formula (Chem.5) hereunder, ##STR00017## in which X is an atom selected from the group comprising oxygen (O) and sulphur (S) and R4 is a hydrocarbon-containing group selected from the group comprising a 3-phytyl group and linear hydrocarbon chains having from 8 to 18 carbon atoms; bromadiolone, brodifacoum, flocoumafen, difenacoum and difethialone.

4. The bait according to claim 1, wherein said composition of AVK(s) comprises difethialone.

5. The bait according to claim 4, wherein said composition of AVK(s) is formed exclusively from difethialone.

6. The bait according to claim 1, wherein said vitamin D composition comprises at least one 9,10-secosteroid compound capable of inducing an increase of tissue calcium.

7. The bait according to claim 1, wherein said vitamin D composition comprises at least one compound selected from the group comprising cholecalciferol, ergocalciferol and calcitriol.

8. The bait according to claim 1, it wherein said bait is formed from said composition of AVK(s), said vitamin D composition and at least one edible excipient that is not rodenticidal for target harmful rodents, and in that: said composition of AVK(s) is formed from difethialone; said vitamin D composition is formed from cholecalciferol; the proportion by weight of difethialone in the rodenticidal bait is between 0.70 ppm and 1.20 ppm, inclusive, and in that the proportion by weight of cholecalciferol in the rodenticidal bait is between 20 ppm and 90 ppm, inclusive.

9. The bait according to claim 1, wherein: said composition of AVK(s) is formed from bromadiolone; said vitamin D composition is formed from cholecalciferol; the proportion by weight of bromadiolone in the rodenticidal bait is between 1.2 ppm and 1.7 ppm, inclusive, and in that the proportion by weight of cholecalciferol in the rodenticidal bait is between 20 ppm and 90 ppm, inclusive.

10. The bait according to claim 1, it wherein said bait is rodenticidal without a significant increase in blood clotting time.

11. A method for controlling a population of target harmful rodents that are resistant to at least one compound that inhibits vitamin K recycling, said method comprising the steps of: Applying a rodenticidal bait according to claim 1.

12. The method according to claim 11, wherein at least one compound that inhibits vitamin K recycling is a compound in the group comprising warfarin, coumatetralyl, dicoumarol, acenocoumarol, phenprocoumon, 3-(1-(4′-fluorobiphenyl-4-yl)-ethyl)-4-hydroxy-2H-1-benzopyran-2-one of formula (Chem.4) hereunder, ##STR00018## compounds of formula (Chem.5) hereunder, ##STR00019## in which X is an atom selected from the group comprising oxygen (O) and sulphur (S) and R4 is a hydrocarbon-containing group selected from the group comprising a 3-phytyl group and linear hydrocarbon chains having from 8 to 18 carbon atoms, bromadiolone and difenacoum.

13. A method of controlling target harmful rodents in which a rodenticidal bait by ingestion is disseminated; so as to be able to be ingested by target harmful rodents for a period, called the consumption time, imposed owing to said ingestion, and in a sufficient amount to be lethal for target harmful rodents; the rodenticidal bait by ingestion comprising: a composition, called composition of AVK(s), of at least one compound that inhibits vitamin K recycling; a composition, called vitamin D composition, of at least one hypercalcaemia-causing vitamin D, and at least one excipient that is edible for target harmful rodents; wherein: said composition of AVK(s) is present in the rodenticidal bait in a proportion by weight, called proportion of AVK(s) by weight, that is not zero and is not lethal on its own for any target harmful rodent, and in that said vitamin D composition is present in the rodenticidal bait in a proportion by weight, called the proportion of vitamin D by weight: sufficient for said vitamin D composition to be lethal on its own for at least one target harmful rodent, and below a minimum proportion by weight sufficient for said vitamin D composition to be lethal on its own for any target harmful rodent.

14. The method according to claim 13, wherein the method is a method for selectively controlling a population of target harmful rodents in which the rodenticidal bait by ingestion is disseminated: so as to be able to be ingested by target harmful rodents of the population of target harmful rodents, and in a sufficient amount to be lethal for females of target harmful rodents consuming this rodenticidal bait, and in which; said composition of AVK(s) and said vitamin D composition of the rodenticidal bait forming a composition of active substance in proportion by weight in the rodenticidal bait selected so that the rodenticidal bait is lethal for a proportion, called female mortality, of females of target harmful rodents consuming this rodenticidal bait and to be lethal for a proportion, called male mortality, of males of target harmful rodents consuming this rodenticidal bait, said female mortality being greater than said male mortality.

Description

[0142] FIG. 1 is a graphical representation illustrating the synergistic effect of a treatment of rodents by gavage with a composition according to the invention combining bromadiolone and cholecalciferol (filled triangle, .box-tangle-solidup.) on warfarin-sensitive rodents, compared to treatment with bromadiolone alone (filled diamond, .diamond-solid.) or cholecalciferol alone (filled circle, .circle-solid.) at the same dose;

[0143] FIG. 2 is a graphical representation illustrating the blood clotting time (Quick's time) of warfarin-sensitive rodents treated with a composition according to the invention combining bromadiolone and cholecalciferol, or treated with bromadiolone alone or treated with cholecalciferol alone as illustrated in [FIG. 1] compared to untreated rodents;

[0144] FIG. 3 is a graphical representation illustrating the synergistic effect of treatment with a composition according to the invention combining difethialone and cholecalciferol (filled triangle, .box-tangle-solidup.) on warfarin-sensitive rodents, compared to treatment with difethialone alone (filled diamond, .diamond-solid.) or cholecalciferol alone (filled circle, .circle-solid.) at the same dose, and

[0145] FIG. 4 is a graphical representation illustrating the synergistic effect of treatment with a composition according to the invention combining difethialone and cholecalciferol (filled triangle, .box-tangle-solidup.) on warfarin-resistant rodents, compared to treatment with difethialone alone (filled diamond, .diamond-solid.) or cholecalciferol alone (filled circle, .circle-solid.) at the same dose.

[0146] A rodenticidal bait by ingestion according to the invention comprises at least one excipient that is edible for target harmful rodents, a composition, called composition of AVK(s), of at least one compound that inhibits vitamin K recycling and a composition, called vitamin D composition, of at least one hypercalcaemia-causing vitamin D. Said composition of AVK(s) is present in the rodenticidal bait according to the invention with a proportion by weight, called proportion of AVK(s), selected so that said composition of AVK(s) is not rodenticidal on its own for each target harmful rodent of the population of rodents whose control is desired. Furthermore, said composition of AVK(s) is not anticoagulating. Said vitamin D composition is present in the rodenticidal bait according to the invention with a proportion by weight, called proportion of vitamin D, selected so that said vitamin D composition is not lethal at 100% on its own. Quite surprisingly and unforeseeably, a rodenticidal bait by ingestion according to the invention is effective on any population of target harmful rodents, in particular on any population of warfarin-sensitive target harmful rodents and on a majority of target harmful rodents resistant to at least one anticoagulant of the antivitamin K type, and in particular resistant to warfarin. The surprising rodenticidal efficacy of a rodenticidal bait by ingestion according to the invention seems to be based on a synergistic effect provided by the combination of said vitamin D composition and said composition of AVK(s), the synergistic effect provided being distinct from an anticoagulant effect. Accordingly, a rodenticidal bait by ingestion according to the invention reduces the risk of secondary poisoning of non-target animals.

[0147] Bromadiolone

[0148] Synergy of Bromadiolone and Cholecalciferol

[0149] A group of 10 warfarin-sensitive male OFA-Sprague-Dawley rats (Charles River, L′Arbresles, France), aged 10 weeks and with a body weight between 160 and 180 g, are fed by gavage for 5 consecutive days with a gavage solution containing bromadiolone (LIPHATECH, France) and cholecalciferol (Sigma-Aldrich, France) in a solvent consisting of 95% (v/v) of oil and 5% (v/v) of DMSO. During this period of gavage, the rats are fed with a conventional solid feed and water in unlimited amounts. The rats are fed by gavage with a volume of this gavage solution so as to administer an amount of bromadiolone corresponding to 0.1 mg of bromadiolone per kilogram of body weight of the rat fed by gavage and an amount of cholecalciferol corresponding to 7.5 mg of cholecalciferol per kilogram of body weight of the rat fed by gavage. The rats in two control groups, each consisting of 10 male OFA-Sprague-Dawley rats, are fed by gavage in the same conditions with gavage solutions containing, respectively, bromadiolone alone so as to administer an amount of bromadiolone corresponding to 0.1 mg of bromadiolone per kilogram of body weight of the rat fed by gavage and cholecalciferol alone so as to administer an amount of cholecalciferol corresponding to 7.5 mg of cholecalciferol per kilogram of body weight of the rat fed by gavage. The percentage of rats surviving gavage is noted daily for 20 days counting from the first day of gavage (D0). The results presented [FIG. 1] show the variation of the percentage of rodents surviving (in %) as a function of time (t, in days) following the first day of gavage. The rats fed by gavage with bromadiolone solution (filled diamond, .diamond-solid.) have 100% survival after 20 days of observation. The rats fed by gavage with the cholecalciferol solution (filled circle, .circle-solid.) have 70% survival after 20 days of observation. Quite surprisingly and unforeseeably, none of the rats fed by gavage with the solution of bromadiolone combined with cholecalciferol (filled triangle, .box-tangle-solidup.) survives for 20 days of observation. The mortality in the group of rats fed by gavage with the solution of bromadiolone combined with cholecalciferol is 100% starting from 7 days after the start of gavage.

[0150] Prothrombin Level—Quick's Time

[0151] Warfarin-sensitive male OFA-Sprague-Dawley rats (Charles River, L′Arbresles, France) are fed by gavage for 3 consecutive days with a gavage solution containing bromadiolone (LIPHATECH, France) and cholecalciferol (Sigma-Aldrich, France) in a solvent consisting of 95% (v/v) of oil and 5% (v/v) of DMSO. During this period of gavage, the rats are fed with a conventional solid feed and water in unlimited amounts. The concentrations of bromadiolone and cholecalciferol in the gavage solution are 0.1 g/kg (100 ppm) and 7.5 g/kg (7500 ppm) respectively. Each rat receives daily a volume of gavage solution calculated to correspond to 1 mL of gavage solution per kilogram of body weight. The OFA-Sprague-Dawley rats in two control groups are fed by gavage in the same conditions with gavage solutions containing bromadiolone (alone) at a concentration of 100 ppm or cholecalciferol (alone) at a concentration of 7500 ppm, respectively. 24 hours after the last gavage, a volume of blood is taken from each rat by intracardiac puncture and placed in a specimen tube containing sodium citrate. The animals are euthanized after puncture and the plasma is prepared by centrifugation. Quick's time (or clotting time) is evaluated on the plasma supplemented with sodium citrate, then decalcified and recalcified in the presence of calcium thromboplastin. Quick's time is evaluated by means of an optical device for clotting analysis (Option 2plus, Bio Mèrieux®). The results are presented [FIG. 2]. The average Quick's time measured on the plasma from the rats fed by gavage with bromadiolone (bar “B”) is 12.4 seconds (±1.8), the average Quick's time measured on the plasma from the rats fed by gavage with cholecalciferol (bar “C”) is 12.4 seconds (±0.9) and the average Quick's time measured on the plasma from the rats fed by gavage with the composition comprising bromadiolone and cholecalciferol (bar “D”) is 14.1 seconds (±2.7). The differences in values of Quick's time measured between the three groups of rats are not significant. The rats in the group of rats that received the combination of bromadiolone and cholecalciferol coagulate in the same way as the rats in the group of rats that received the gavage solution containing bromadiolone on the one hand and the rats in the group of rats that received the gavage solution containing cholecalciferol on the other hand. Quick's time for rats treated with a control solution formed from oil (95%, v/v) and DMSO (5%, v/v) is (bar “A”) 12.8 seconds (±2.3). Neither the gavage solution containing bromadiolone in combination with cholecalciferol, nor the gavage solution containing bromadiolone alone, nor the gavage solution containing cholecalciferol induces an increase of Quick's time relative to the control. They are not anticoagulants. The gavage solution containing bromadiolone and cholecalciferol in combination according to the invention is a rodenticide and is not an anticoagulant.

[0152] Rodenticidal Bait by Ingestion According to the Invention Tested on L120Q Sprague-Dawley Rats that have Undergone Introgression

[0153] L120Q Sprague-Dawley (“SD”) rats (10 male rats and 10 female rats) that have undergone introgression, which are homozygotic carriers of the L120Q mutation in the Vkorc1 gene, are acclimatized for 5 days in individual cages in the presence of a conventional solid feed and water in unlimited amounts. At D0, the conventional solid feed is removed and replaced with a rodenticidal bait by ingestion according to the invention in a sufficient amount to satisfy the rodents' appetite and without any other choice of consumption (“no choice”) than the rodenticidal bait alone. The rodenticidal bait by ingestion according to the invention is made available for 5 days, and then is replaced with a conventional solid feed. The rodenticidal bait by ingestion according to the invention is made up of flour of cereals, fat, starch, a red dye and an oily solvent as edible excipient, bromadiolone in a proportion by weight of 0.9 ppm and cholecalciferol in a proportion by weight of 75 ppm. The duration of monitoring is 21 days counting from the first day of presentation of the baits (D0). The rats stop feeding after 3 days of consumption of the rodenticidal bait. The mortality and the average delay before death of the animal are given in Table 1 hereunder for the male rats and for the female rats.

TABLE-US-00001 TABLE 1 “SD” having undergone introgression L120Q Mortality, % Delay (days) Male 66 6.5 Female 100 4.5

[0154] The average mortality (males and females) of the L120Q “SD” rats that had undergone introgression is of the order of 83%. The rodenticidal bait by ingestion according to the invention is more effective on these female rats than on these male rats. In any case, no haemorrhagic sign is observed in these rats (males and females) that had consumed the rodenticidal bait by ingestion according to the invention.

[0155] Rodenticidal Bait by Ingestion According to the Invention Tested on House Mice (“Mus musculus”) Bearing the L128S Mutation

[0156] A test carried out in similar conditions to the test carried out with L120Q “SD” rats that had undergone introgression described above is carried out with male and female house mice (Mus musculus) that are homozygotic carriers of the L128S mutation in the Vkorc1 gene. The rodenticidal bait by ingestion according to the invention is made up of flour of cereals, fat, starch, a red dye and an oily solvent as edible excipient, bromadiolone in a proportion by weight of 1 ppm and cholecalciferol in a proportion by weight of 75 ppm. The duration of monitoring is 21 days counting from the first day of presentation of the baits (D0). The mice stop feeding after 3 days of consumption of the rodenticidal bait. The mortality and the average delay before death of the animal are given in Table 2 hereunder.

TABLE-US-00002 TABLE 2 L128S mice Mortality, % Delay (days) Male 30 9.5 Female 70 5.5

[0157] The rodenticidal bait by ingestion according to the invention is more effective on the female house mice bearing the L128S mutation than on the male mice bearing the L128S mutation. In any case, no haemorrhagic sign is observed in the house mice bearing the L128S mutation (males and females) that consumed the rodenticidal bait by ingestion according to the invention.

[0158] Rodenticidal Bait by Ingestion According to the Invention on “Spretus” House Mice Bearing the Vkorc1 Gene

[0159] A test carried out in similar conditions to the test carried out with L120Q “SD” rats that had undergone introgression, described above, is carried out with “Spretus” house mice bearing the Vkorc1 gene. The rodenticidal bait by ingestion according to the invention is made up of flour of cereals, fat, starch, a red dye and an oily solvent as edible excipient, bromadiolone in a proportion by weight of 1 ppm and cholecalciferol in a proportion by weight of 75 ppm. The duration of monitoring is 21 days counting from the first day of presentation of the baits (D0). The mice stop feeding after 3 days of consumption of the rodenticidal bait. The mortality and the average delay before death of the animal are given in Table 3 hereunder.

TABLE-US-00003 TABLE 3 “Spretus” mice Mortality, % Delay (days) Male 10 7 Female 30 10

[0160] The rodenticidal bait by ingestion according to the invention is more effective on the female “Spretus” mice than on the male “Spretus” mice. In any case, no haemorrhagic sign is observed in the male and female “Spretus” mice that consumed the rodenticidal bait by ingestion according to the invention.

[0161] Relative Toxicity of Cholecalciferol in Male Rodents and Female Rodents

[0162] Warfarin-sensitive Sprague-Dawley rats (20 male rats and 20 female rats), male and female rats that are homozygotic carriers of the L120Q mutation, male and female Sprague-Dawley rats that are homozygotic carriers of the Y139F mutation and male and female brown rats (Rattus norvegicus) that are homozygotic carriers of the Y1 39C mutation are acclimatized for 5 days in the presence of a conventional solid feed and water in unlimited amounts. The rats are fed by gavage with a single dose of a gavage solution containing cholecalciferol (Sigma-Aldrich, France) in a solvent consisting of 95% (v/v) of oil and 5% (v/v) of DMSO. The rats are fed with a conventional solid feed and water in unlimited amounts. The amount of cholecalciferol administered is 30 mg of cholecalciferol per kilogram of body weight of the rat fed by gavage. The percentage of rats surviving gavage for 20 days, counting from the first day of gavage, is noted daily. The results given in Table 4 hereunder describe the mortality observed (“SD”, Sprague-Dawley). The amount of cholecalciferol ingested by the rodents during gavage corresponds to consumption by these rodents of a bait assayed at 300 ppm of cholecalciferol.

TABLE-US-00004 TABLE 4 Rodents Male mortality, % Female mortality, % “SD” 10 30 L120Q 15 5 Y139F 30 0 Y139C 30 0

[0163] The use of cholecalciferol 300 ppm for controlling warfarin-resistant target harmful rodents leads to selection of the female warfarin-resistant rodents whereas the use of a rodenticidal bait by ingestion according to the invention allows on the contrary (Table [1], Table [2] and Table [3]) selective control directed preferentially at female warfarin-resistant target harmful rodents. This results in a decrease in the number of litters of target harmful rodents and improved efficacy of control.

[0164] Difethialone

[0165] Absence of Rodenticidal Effect of a Control Bait Comprising Difethialone 1 ppm on OFA-Sprague-Dawley Rats

[0166] In a rodenticidal bait by ingestion according to the invention, said composition of AVK(s) (in this test, difethialone) is present in the rodenticidal bait in a proportion by weight, called proportion of AVK(s) by weight, that is not zero (1 ppm) and is not lethal on its own for any target harmful rodent consuming a bait, called bait of AVK(s) (difethialone bait), formed from said at least one excipient and said composition of AVK(s) (difethialone)—alone as active substance in said bait of AVK(s)—and in said proportion of AVK(s) (1 ppm) in said bait of AVK(s). Warfarin-sensitive OFA-Sprague-Dawley white rats (5 male and 5 female) are put in individual cages and are acclimatized for 3 days in these cages and fed with a conventional solid feed and water in unlimited amounts. At D0, the conventional solid feed is replaced with a ration of bait, called difethialone bait, comprising difethialone in a proportion by weight of 1 ppm (1 mg of difethialone per kilogram of bait). The ration of difethialone bait supplied is 50 grams per rat. At D1, D2 and D3, the amount of difethialone bait consumed by each rat is measured and then the ration of difethialone bait is made up to 50 grams. Each animal is weighed at D0, D1, D2, D3 and D4. The average consumption of difethialone bait is given in Table 5 hereunder. The rats are kept under surveillance and are fed starting from D4 with a conventional solid feed and water in unlimited amounts until D21. No refusal of food is observed. 100% of the rats survive to D21. None of the rats presents a haemorrhagic sign. Said difethialone bait at 1 ppm is not rodenticidal for OFA-Sprague-Dawley rats consuming this bait for 3 days.

TABLE-US-00005 TABLE 5 Average consumption, g/kg of rat's weight D 1 62.8 D 2 71.3 D 3 60.3 D 4 61.6

[0167] Synergy of Difethialone and Cholecalciferol on Warfarin-Sensitive Rats

[0168] A group of warfarin-sensitive male OFA-Sprague-Dawley rats (Charles River, L′Arbresles, France), aged 10 weeks and with a body weight between 160 and 180 g, are fed by gavage once a day for 3 consecutive days with a gavage solution containing difethialone (LIPHATECH, France) and cholecalciferol (Sigma-Aldrich, France) in a solvent consisting of 95% (v/v) of oil and 5% (v/v) of DMSO. During this period of gavage, the rats are fed with a conventional solid feed and water in unlimited amounts. The amounts of difethialone and cholecalciferol administered are 125 μg per kilogram of body weight of the rat fed by gavage and 7.5 mg per kilogram of body weight of the rat fed by gavage, respectively. The rats in two control groups, each consisting of OFA-Sprague-Dawley rats, are fed by gavage in the same conditions with gavage solutions containing difethialone alone (125 μg/kg) or cholecalciferol alone (7.5 mg/kg), respectively. The percentage of rats surviving gavage is recorded daily, for 20 days counting from the first day of gavage. The results presented [FIG. 3] show the variation of the percentage of rodents surviving (in %) as a function of time (t, in days) following the first day of gavage. The rats fed by gavage with the difethialone solution (.diamond-solid.) have 100% survival after 20 days of observation. The rats fed by gavage with the cholecalciferol solution (.circle-solid.) show 60% survival after 2 days of observation. Quite surprisingly and unforeseeably, none of the rats fed by gavage with the solution of difethialone combined with cholecalciferol (.box-tangle-solidup.) survives for 20 days of observation. The mortality in the group of rats fed by gavage with the solution of difethialone combined with cholecalciferol is 100% starting from 8 days after the start of gavage. The synergistic effect has been demonstrated.

[0169] Dose/Rodenticidal Response Effect of Difethialone on Warfarin-Sensitive Rats

[0170] A gavage solution containing difethialone (LIPHATECH, France) in a solvent consisting of 95% (v/v) of oil and 5% (v/v) of DMSO is administered once a day for 3 consecutive days to warfarin-sensitive male OFA-Sprague-Dawley rats (Charles River, L′Arbresles, France), aged 10 weeks and with a body weight between 160 and 180 g. During this period of gavage, the rats are fed with a conventional solid feed and water in unlimited amounts. The doses of difethialone administered to the OFA-Sprague-Dawley rats, given in Table 6 hereunder, are expressed in micrograms of difethialone administered per kilogram of body weight of the gavage-fed rodent.

TABLE-US-00006 TABLE 6 Difethialone dose, μg/kg Mortality, % 0 0 50 0 100 0 150 40 200 100 500 100

[0171] The percentage of rats surviving gavage is recorded daily for 21 days counting from the first day of gavage.

[0172] Prothrombin Level—Quick's Time

[0173] Nine warfarin-sensitive male OFA-Sprague-Dawley rats (Charles River, L′Arbresles, France) are fed by gavage for 3 consecutive days with difethialone (LIPHATECH, France) in solution in a mixture consisting of 90% (v/v) of oil and 10% (v/v) of DMSO. The concentration of difethialone in the gavage solution is 0.1 g/kg (100 ppm). Each rat receives daily a volume of gavage solution calculated to correspond to 1 mL of gavage solution per kilogram of its body weight (i.e. 1 ppm). 24 hours after the last gavage, a volume of blood is taken from each rat by intracardiac puncture and placed in a specimen tube containing sodium citrate. The animals are euthanized after puncture and the plasma is prepared by centrifugation. No haemorrhagic sign is observed in the rats fed by gavage with the difethialone solution. Quick's time (or clotting time) is evaluated on the plasma supplemented with sodium citrate, then decalcified and recalcified in the presence of calcium thromboplastin. Quick's time is evaluated by means of an optical device for clotting analysis (Option 2plus, Bio Mèrieux®). The average Quick's time measured on the plasma from the rats fed by gavage with difethialone is 23.4 seconds (±6.1). For comparison, the average Quick's time of control rats fed by gavage with the solution without difethialone is 17.5 seconds (±2.1). The difference in values of Quick's time of the rats fed by gavage with the difethialone solution and with the control solution without difethialone is not significant. The value of Quick's time of the rats fed by gavage with the difethialone solution, not significantly increased relative to the Quick's time of the rats in the control group, is representative of a normal coagulation situation, the blood of the rats that had received the gavage solution comprising difethialone clotting comparably to the blood of the control rats. The gavage solution containing difethialone alone does not induce an increase in Quick's time relative to the control and is not anticoagulating.

[0174] Dose/Rodenticidal Response Effect of Cholecalciferol

[0175] A gavage solution containing cholecalciferol (Sigma-Aldrich, France) in a solvent consisting of 95% (v/v) of oil and 5% (v/v) of DMSO is administered once a day for 3 consecutive days to warfarin-sensitive male OFA-Sprague-Dawley rats (Charles River, L′Arbresles, France), aged 10 weeks and with a body weight between 160 and 180 g. During this period of gavage, the rats are fed with a conventional solid feed and water in unlimited amounts. The doses of cholecalciferol administered to the OFA-Sprague-Dawley rats, given in Table 7 hereunder, are expressed in milligrams of cholecalciferol administered per kilogram of body weight of the gavage-fed rodent.

TABLE-US-00007 TABLE 7 Cholecalciferol dose, mg/kg Mortality, % 0 0 5 20 7.5 30

[0176] The percentage of rats surviving gavage is noted daily for 21 days counting from the first day of gavage.

[0177] Synergy of Difethialone and Cholecalciferol on Warfarin-Resistant Female Sprague-Dawley Y139F Rats

[0178] Female Sprague-Dawley rats that are homozygotic carriers of the Y139F mutation in the Vkorc1 gene, aged 10 weeks, are acclimatized for 5 days in the presence of a conventional solid feed and water in unlimited amounts. These female Sprague-Dawley Y139F rats are fed by gavage once a day for 3 consecutive days with a gavage solution containing difethialone (LIPHATECH, France) and cholecalciferol (Sigma-Aldrich, France) in a solvent consisting of 95% (v/v) of oil and 5% (v/v) of DMSO. During this period of gavage, the rats are fed with a conventional solid feed and water in unlimited amounts. The amounts of difethialone and cholecalciferol administered are 0.125 mg per kilogram of body weight of the rat fed by gavage and 7.5 mg per kilogram of body weight of the rat fed by gavage, respectively. The rats in two control groups, each consisting of OFA-Sprague-Dawley Y137F rats, are fed by gavage in the same conditions with gavage solutions containing, respectively, difethialone alone so as to administer an amount of difethialone corresponding to 0.125 mg per kilogram of body weight of the rat fed by gavage and cholecalciferol alone so as to administer an amount of cholecalciferol corresponding to 7.5 mg per kilogram of body weight of the rat fed by gavage. The percentage of rats surviving gavage is noted daily for 21 days counting from the first day of gavage. The results presented [FIG. 4] show the variation of the percentage of rodents surviving (in %) as a function of time (t, in days) following the first day of gavage. The rats fed by gavage with the difethialone solution (.diamond-solid.) have 100% survival after 22 days of observation. The rats fed by gavage with the cholecalciferol solution (.circle-solid.) show 90% survival after 22 days of observation. Quite surprisingly and unforeseeably, none of the rats fed by gavage with the solution of difethialone combined with cholecalciferol (.box-tangle-solidup.) survives to 22 days of observation. The mortality in the group of rats fed by gavage with the solution of difethialone combined with cholecalciferol is 100% starting from 9 days after the start of gavage. The synergistic effect has been demonstrated.

[0179] Synergy of Difethialone and Cholecalciferol on Male and Female Sprague-Dawley Rats, L120Q, Y139F and Y139C

[0180] Warfarin-sensitive male and female Sprague-Dawley rats, male and female rats that are homozygotic carriers of the L120Q mutation, male and female Sprague-Dawley rats that are homozygotic carriers of the Y139F mutation and male and female brown rats (Rattus norvegicus) that are homozygotic carriers of the Y139C mutation are acclimatized for 5 days in the presence of a conventional solid feed and water in unlimited amounts. The rats are fed by gavage once a day for 3 consecutive days with a gavage solution containing difethialone (LIPHATECH, France) and cholecalciferol (Sigma-Aldrich, France) in a solvent consisting of 95% (v/v) of oil and 5% (v/v) of DMSO. During this period of gavage, the rats are fed with a conventional solid feed and water in unlimited amounts. The amounts of difethialone and cholecalciferol administered are 0.125 mg per kilogram of body weight of the rat fed by gavage and 7.5 mg per kilogram of body weight of the rat fed by gavage, respectively. The percentage of rats surviving gavage is noted daily for 21 days counting from the first day of gavage. The results given in Table 8 describe the mortality and the mean time to occurrence (“MTO”) of this mortality (“SD”, Sprague-Dawley).

TABLE-US-00008 TABLE 8 Male MTO, Female Line Mortality, % days Mortality, % MTO “SD” 100 6.8 n.d. n.d. L120Q 100 5.3 100 5.1 Y139F 100 6.1 100 5.5 Y139C 100 5.4 100 6.7

[0181] Dose/Rodenticidal Response Effect of Difethialone on Warfarin-Resistant Male Y139F Rats

[0182] A gavage solution containing difethialone (LIPHATECH, France) in a solvent consisting of 95% (v/v) of oil and 5% (v/v) of DMSO is administered once a day for 3 consecutive days to male Sprague-Dawley rats that have undergone introgression, which are homozygotic carriers of the Y139F mutation in the Vkorc1 gene. During this period of gavage, the rats are fed with a conventional solid feed and water in unlimited amounts. The doses of difethialone administered to the Sprague-Dawley rats that have undergone introgression, given in Table 9 hereunder, are expressed in micrograms of difethialone administered per kilogram of body weight of the gavage-fed rodent.

TABLE-US-00009 TABLE 9 Difethialone dose, μg/kg Mortality, % 0 0 100 0 125 0 250 100 500 100

[0183] The percentage of rats surviving gavage is noted daily for 21 days counting from the first day of gavage. 100% of the Sprague-Dawley rats that had undergone introgression survive gavage for 3 days with 0.125 mg of difethialone per kilogram of body weight per day.

[0184] The invention may be the subject of many variants and applications other than those described above. In particular, it goes without saying that unless stated otherwise, the various structural and functional features of each of the embodiments described above are not to be regarded as combined and/or closely and/or inextricably linked together, but on the contrary as simple juxtapositions. Furthermore, the structural and/or functional features of the different embodiments described above may be the subject wholly or partly of any different juxtaposition or of any different combination.