NZ548082A - Dermal pesticide containing vitamin D3 - Google Patents

Abstract

Disclosed is a method of killing a non-human animal pest comprising topically administering to the animal pest a topical pesticide formulation comprising an effective amount of cholecalciferol or 25-hydroxycholecalciferol and at least one carrier which is capable of delivering the cholecalciferol or 25-hydroxycholecalciferol transdermally to cause the death of an animal pest. Also disclosed is an apparatus for delivering a topical pesticide formulation to an animal pest, said apparatus comprising means forming at least one partially enclosed space into which the animal can enter or pass through, and at least one automatic dispensing apparatus having a spray means, and a container containing a formulation comprising an effective amount of cholecalciferol or 25-hydroxycholecalciferol and at least one carrier which is capable of delivering the cholecalciferol or 25-hydroxycholecalciferol transdermally to the animal, said automatic dispensing apparatus being operatively connected to the enclosed space, said enclosed space including at least one sensing means therein to detect when an animal is present in or near the space, wherein said sensing means activates the automatic dispensing apparatus to deliver a predetermined quantity of the cholecalciferol or 25-hydroxycholecalciferol formulation topically to the animal.

Description

New Zealand Paient Spedficaiion for Paient Number 548082 Patents Form # 5 A 8 0 8 .2 *10056650881* NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION AFTER PROVISIONAL NO: 548082/555104 DATED: 23 June 2006 TITLE: PESTICIDE FORMULATIONS I, AGNEW, Warren Roy Address: 2 Young Street, R D 2, Warkworth, New Zealand Nationality: A New Zealand citizen do hereby declare the invention for which I pray that a patent may be granted to me and the method by which it is to be performed, to be particularly described in and by the following statement: 191143NZ_Cap_20070625J153_SEP.doc FEE CODE 1050 otoeJ 2 5 JUN 200? I ^MCglVBDI 2 Field of Invention The invention generally relates to formulations and methods for animal pest control. Background of Invention Pest control is a major problem in most countries of the world, and particularly in New 5 Zealand due to the wide variety of species, particularly mammals, which were introduced to New Zealand by the early European settlers. Pests such as rabbits, rats, mice, possums, stoats, ferrets and weasels have had a significant effect on native flora and fauna. Rodents (rats and mice) and mustelids (ferrets, stoats and weasels) are significant conservation pests because they are predators and competitors of native fauna. Rats eat bird eggs, chicks and 10 invertebrates as well as fruits and seeds. Predation by rodents is a significant threat to endangered and iconic bird species such as the kokako. Scientific studies have identified stoats as a major predator of bird eggs and chicks, including kiwi. Stoats have large territories and can cover 8 km in a night as they seek food. Without predator control, only about 5% of kiwi chicks survive in the wild. Feral cats, goats, deer and wallabies are other 15 examples of pest species. Due to the damage caused to native flora and fauna, it is necessary to implement measures to control these pests.

There has been steady development and refinement of pest control technologies and approaches in New Zealand over the past 50 years. These have been driven by the need for more specific targeting of pests, more humane methods of killing the pests, greater 20 operational efficiencies and effectiveness. The desired outcomes have not focused on the number of pests killed, but on saving threatened species and ecosystems as well as improving the productivity and health of the primary production sectors, notably agriculture.

Methods of pest control which have been employed in New Zealand and other countries to date include traps and poisons.

Traps have been used to catch possums for many decades. They have also been used to trap stoats, however they have been found to be inefficient. For example, the Department of Conservation in New Zealand is responsible for the setting of some 60,000 stoat traps each and every day of the year. These traps are generally metal and spring loaded, and because of their danger to the ground feeding kiwi, are placed in a box with a restricted opening 191143NZ Complete Specification 20090602.doc 3 designed to keep kiwi away from the traps. However the kill rate from these traps only averages one stoat for every 300 trap nights. During this time the traps have to be checked, re-baited or cleared. Hence a huge amount of expense and effort is being channelled into killing a very cunning and wary animal.

There are six poisons currently registered in New Zealand for possum control: 1080 (sodium fluoroacetate), cyanide, phosphorus, pindone, brodifacoum, and cholecalciferol or 25-hydroxycholecalciferol (Vitamin D3). Some are also registered for the control of other pests. All of these poisons are available in bait form such as pellets, pastes, gels, coated baits, and cereal baits intended for oral ingestion by the target species. Although most of these poisons 10 are effective at killing pests, they have disadvantages. Phosphorus, 1080 and brodifacoum have high secondary poisoning risks to dogs, birds and other animals due to the movement down the food chain and their persistence in the environment. Phosphorus, brodifacoum and pindone are considered to be inhumane methods of pest control generally because it takes a long amount of time before death occurs (for example, a few weeks). Although cyanide is an 15 effective poison and has low environmental persistence and secondary poisoning risk, it is hazardous in paste form and is toxic to humans, requiring a licence to store, handle and use it.

Cholecalciferol or 25-hydroxycholecalciferol, also known as Vitamin D3, is a relatively new poison, introduced in New Zealand in 1995. It was introduced initially for possum control, 20 and is also a rodenticide. It is commercially available in bait form, both as a cereal based bait for killing possums (CAMPAIGN containing 0.8% cholecalciferol or 25-hydroxycholecalciferol), and a paste bait for possums and rats and mice (FERACOL, 0.8% for possums and 0.08% for rats and mice).

In order to gain biological and toxicological activity, cholecalciferol must undergo metabolic 25 conversion to 25-hydroxycholecalciferol. At toxic doses, this active metabolite mobilises calcium stores from the bones into the bloodstream, and decreases calcium excretion by the kidneys. The net result is dangerously high concentrations of blood calcium (hypercalcaemia) and tissue calcification. Death usually results from heart failure.

Although cholecalciferol or 25-hydroxycholecalciferol is effective at killing some species 30 through administration in poisonous bait form, it is not effective in other species. For example, it is not effective in killing members of the mustelid family, such as ferrets, stoats 191143NZ Complete Specification 20090602.doc 4 and weasels due the fact that these animals will not eat poisonous baits. Both stoats and ferrets like to explore tunnels and holes in the search for food and prefer warm salty live kills. These animals are among the most destructive pests due to their predation on native fauna such as the kiwi.

Other disadvantages of poisonous baits is the need to have the bait stations very carefully positioned and administered as the baits may be toxic to non-targeted animals such as dogs, cats, stock and humans.

Object of Invention It is an object of the present invention to provide a formulation and method for animal pest 10 control which ameliorates some of the disadvantages and limitations of the known art or which at least provides the public with a useful choice.

Summary of Invention In a first aspect the invention provides a method of killing a non-human animal pest comprising topically administering to the animal pest a topical pesticide formulation 15 comprising an effective amount of cholecalciferol or 25-hydroxycholecalciferol and at least one carrier which is capable of delivering the cholecalciferol or 25-hydroxycholecalciferol transdermally to cause the death of an animal pest.

Preferably the topical pesticide formulation comprises 4 to 50% w/v of cholecalciferol or 25-hydroxycholecalciferol.

Preferably the cholecalciferol or 25-hydroxycholecalciferol is present in an amount of between about 10 to 40% w/v.

Preferably the cholecalciferol or 25-hydroxycholecalciferol is present in an amount of between about 30 to 40% w/v.

Preferably the formulation is topically administered to the animal by way of an automatic 25 dispensing apparatus including at least one sensing means to detect when the animal is present.

Preferably the formulation is sprayed on to the animal. 19U43NZ Complete Specification 20090602.doc In a yet further aspect the invention provides an apparatus for delivering a topical pesticide wherein said automatic dispensing apparatus has means forming at least one partially enclosed space into which the animal can enter or pass through, and at least one dispenser containing a formulation comprising an effective amount of cholecalciferol or 25-5 hydroxycholecalciferol and at least one carrier which is capable of delivering the cholecalciferol or 25-hydroxycholecalciferol transdermally to the animal, said automatic dispensing apparatus being operatively connected to the enclosed space, said enclosed space including at least one sensing means therein to detect when an animal is present in or near the space, wherein said sensing means activates the automatic dispensing apparatus to spray 10 a predetermined quantity of the cholecalciferol or 25-hydroxycholecalciferol formulation topically to the animal.

Preferably the cholecalciferol or 25-hydroxycholecalciferol formulation is sprayed onto the animal from a pressurised aerosol container.

Preferably the predetermined quantity of the cholecalciferol or 25-hydroxycholecalciferol 15 formulation is sprayed onto a small area of the animal.

Preferably the small area of the animal is a spot of about 2cm in diameter.

Preferably the formulation is administered to the animal in an amount of between about 0.5 -2.0 ml.

Preferably the animal pest is a mouse, rat, stoat, weasel, ferret, rabbit or hedgehog.

In another aspect the invention provides an apparatus for delivering a topical pesticide formulation to an animal pest, said apparatus comprising means forming at least one partially enclosed space into which the animal can enter or pass through, and at least one automatic dispensing apparatus having a spray means, and a container containing a formulation comprising an effective amount of cholecalciferol or 25-hydroxycholecalciferol in a solution 25 which is capable of delivering the cholecalciferol or 25-hydroxycholecalciferol transdermally to the animal, said automatic dispensing apparatus being operatively connected to the enclosed space, said enclosed space including at least one sensing means therein to detect when an animal is present in or near the space, wherein said sensing means 191143NZ Complete Specification 20090602.doc 6 activates the automatic dispensing apparatus to spray a predetermined quantity of the cholecalciferol or 25-hydroxycholecalciferol formulation topically to the animal.

Preferably the spray means and the container comprise a pressurised aerosol container.

Preferably the predetermined quantity of cholecalciferol or 25-hydroxycholecalciferol 5 formulation is between about 0.5 - 2.0 ml.

Preferably the formulation in the container comprises about 4 to 40% w/v of cholecalciferol or 25-hydroxycholecalciferol.

Preferably the formulation in the container comprises about 10 to 40% w/v of cholecalciferol or 25-hydroxycholecalciferol.

Preferably the container contains a formulation comprising from 30 to 40% w/v of cholecalciferol or 25-hydroxycholecalciferol in solution.

In another aspect the invention provides a topical pesticidal formulation comprising about 4 - 50% w/v of cholecalciferol or 25-hydroxycholecalciferol and at least one carrier which is capable of delivering the cholecalciferol or 25-hydroxycholecalciferol transdermally to cause 15 the death of an animal pest.

Preferably the cholecalciferol or 25-hydroxycholecalciferol is present in an amount of between about 10 - 40% w/v.

Preferably the cholecalciferol or 25-hydroxycholecalciferol is present in an amount of between about 30 - 40% w/v.

Preferably the carrier or each carrier is selected from the group comprising absolute alcohol, ethanol, isopropanol, methanol, benzyl alcohol, glycol ethers, glycerol formal, polyethylene glycols, liquid polyoxyethylene glycols, propylene glycol, pyrrolidones such as N-methylpyrrolidone and 2-pyrrolidone, acetone, acetonitrile, butyl diglycol, dimethyl sulfoxides, dimethylacetamide, dimethylformamide, monomethylacetamide, diethyl 25 phthalate, or any combination thereof.

Preferably the carrier or one of the carriers is absolute alcohol.

Preferably the formulation is in the form of a foam, gel, or liquid spray. 191143NZ Complete Specification 20090602.doc 7 Preferably the formulation further includes calcium.

Preferably the formulation is contained within a pressurised aerosol container.

Detailed Description The following description will describe the invention in relation to preferred embodiments of 5 the invention. The invention is in no way limited to these preferred embodiments as they are purely to exemplify the invention only and possible variations and modifications that would be readily apparent without departing from the scope of the invention are intended to be included within the scope of the invention.

The invention relates to a formulation for topical administration to animal pests, wherein the 10 formulation comprises a solution including an effective amount of at least one toxic substance and at least one dermal penetrant. It is believed that an effective lethal dose of the toxic substance can be dermally delivered to an animal pest in solution, thereby being absorbed into the animal's bloodstream and subsequently causing the death of the animal.

As used herein, the term "toxic substance" is intended to cover cholecalciferol or 25-15 hydroxycholecalciferol alone or in combination with any other poisonous substance capable of causing the death of an animal.

For example, the additional toxic substances which may be useable in the invention include but are not limited to the following: sodium fluoroacetate, cyanide, phosphorus, pindone, brodifacoum, nicotine, p-aminopropiophenone, and derivatives or metabolites thereof.

As used herein, the term "dermal penetrant" is intended to include all substances or materials which are capable of penetrating the skin into the systemic circulation and/or of moving molecules from the outer surface of the skin into the systemic circulation. The dermal penetrant is typically a liquid solvent which facilitates or enables the transdermal delivery of the toxic substance to the animal pest.

The dermal penetrant or solvent used in the formulation should be chosen for its ability to enhance dermal absorption, bioavailability and subsequent toxicity of the toxic substance.

Suitable dermal penetrants include, for example, absolute alcohol, ethanol, isopropanol, methanol, benzyl alcohol, glycol ethers, glycerol formal, polyethylene glycols, liquid 191143NZ Complete Specification 20090602.doc 8 polyoxyethylene glycols, propylene glycol, pyrrolidones such as N-methylpyrrolidone and 2-pyrrolidone, acetone, acetonitrile, butyl diglycol, dimethyl sulfoxides, dimethylacetamide, dimethylformamide, monomethylacetamide, diethyl phthalate, or any combination thereof.

As used herein, the term "effective amount" refers to an amount effective to achieve the 5 desired effect, that is, the death of the animal pest.

The amount of toxic substance present in the formulation typically depends on the species of animal pest to be treated and the size of the animal pest. The amount of toxic substance present is ideally determined by the amount (mg/kg) of the toxic substance needed to meet the LD50 of the target species (that is, the lethal dose required to kill 50% of animals). 10 Dermal toxicity (LD50) values will vary depending on the type of toxic substance used and the target species. Values often also vaiy within the. species, depending on the sex of the animal.

It is envisaged that silicone may be added to the formulation to assist with adhesion of the formulation to the fur or skin of the animal pest in cases where the fur or skin might be wet.

Preferred Embodiments In one preferred embodiment of the invention the toxic substance is cholecalciferol or 25-hydroxycholecalciferol, and the dermal penetrant is absolute alcohol.

Cholecalciferol or 25-hydroxycholecalciferol is commercially available in two forms, that is, as a white powder and a solid resin block. Formulations of the present invention have been 20 prepared containing cholecalciferol or 25-hydroxycholecalciferol as the toxic substance and absolute alcohol as the dermal penetrant. However other dermal penetrants or mixtures thereof may be used in the invention.

The formulations have been prepared using cholecalciferol or 25-hydroxycholecalciferol in resin block form. The resin block is heated until it melts (usually at a temperature of 25 between 50 - 90°C). It is then dissolved in oil and remains in solution as it cools. It is envisaged that the powder form of cholecalciferol or 25-hydroxycholecalciferol could also be used to make the formulations of the invention. 191143NZ Complete Specification 20090602.doc 9 A solution of the required amount of cholecalciferol or 25-hydroxycholecalciferol and the absolute alcohol or other dermal penetrant(s) is then prepared. The amount of cholecalciferol or 25-hydroxycholecalciferol included in the formulation varies depending on the target pest species and the size of the pest species.

Preferably, the cholecalciferol or 25-hydroxycholecalciferol is present in an amount of between about 4 - 50% w/v. More preferably, the cholecalciferol or 25-hydroxycholecalciferol is present in an amount of between about 10 - 40% w/v.

It is envisaged that calcium may be added to the formulation to improve the efficacy of the formulation.

The formulation is designed to be topically administered to the animal pest. The formulation may be in the form of a foam, gel or liquid spray.

Preferably the formulation is applied to the animal by way of an eradication apparatus as described by the present inventor in New Zealand Patent No. 540012. The eradication apparatus includes a tunnel and an automatic dispensing apparatus in the form of a spray 15 means such as a pressurised aerosol container containing the formulation. The automatic dispensing apparatus is operatively connected to the tunnel, together with at least one sensing means so that when an animal passes through the tunnel, the sensors detect the presence of the animal and sense the body length or size of the animal and activate the spray means to deliver a pre-determined amount of the formulation to the fur or skin surface of the target 20 animal, which will be absorbed into the animal's system and cause its death. Such an eradication apparatus is advantageous for killing pests such as mustelids, as all mustelids run through tunnels due to their natural curiosity, so no lure is required. It is envisaged that the eradication apparatus could be modified depending on the type of animal pest to be targeted. For example, instead of tunnels, sections of large diameter (200-300 mm) pipes could be 25 used fitted with the sensor activated pump or spray units to apply the toxin. The target species may use such pipes for shelter, hiding or resting.

Other spray means in the form of pump devices or the like may be used. For example, pumps may be more effective at delivering the formulation in cold climates where there is not enough pressure to deliver the formulation from a pressurised aerosol container. 191143NZ Complete Specification 20090602.doc Preferably, the formulation is administered in an amount of between about 0.5 - 2.0 ml per animal.

Dosage rates typically depend on the species of animal pest and the size of the animal pest. Small animals move very fast so it may only be possible to topically apply 0.5 ml of the 5 formulation via the eradication apparatus if the animal runs through the apparatus quickly, in which case a higher concentration formulation could be applied to ensure that an effective amount of the toxic substance is delivered to the animal's bloodstream.

A formulation of the present invention was initially prepared and tested on mice and laboratory rats to determine the viability of the invention. The formulation contained 10 cholecalciferol or 25-hydroxycholecalciferol in an amount of 4% w/v and absolute alcohol. The formulation was applied to the mice and laboratory rats in an amount of 0.5 ml per animal, and in each case death of the animal occurred within a reasonably short time frame. Given these results, further studies were carried out to test the efficacy of the formulations of the invention.

Example 1 Two formulations of the invention were prepared both containing cholecalciferol or 25-hydroxycholecalciferol in solution with absolute alcohol. One of the formulations contained cholecalciferol or 25-hydroxycholecalciferol in an amount of 20% w/v. The other formulation contained cholecalciferol or 25-hydroxycholecalciferol in an amount of 40% 20 w/v. • The formulation was topically applied to several animal pest species in varying amounts and the animals were studied. The results of the study are set out in Table 1 below.

Table 1. Results of dermal application of cholecalciferol or 25-hydroxycholecalciferol in various animal pest species.

Animal Species Weight of Animal Amount of formulation applied % cholecalciferol or 25- hydroxycholecalciferol in solution Observations Lab Rat Unknown 0.5 ml % Death occurred 191143NZ Complete Specification 20090602.doc 11 (Rattis norvegicus) within 72 hours Bush Rat (Rattis rattis) Unknown 1.0 ml % Death occurred within 40 hours Rabbit 2.6 kg 2.0 ml % Death occurred within 2 days Hedgehog 515 g 1.5 ml % Death occurred within 25 hours Weasel 139 g 1.5 ml 40% Death occurred within 36 hours Weasel 116 g 1.5 ml 40% Death occurred within 43 hours Ferret 902 g 2.0 ml 40% Death occurred within 48 hours It was observed that death appeared to occur without causing pain to the animal.

Example 2 Methodology A test solution of approximately 100 ml was formulated, containing Vitamin D3 (cholecalciferol or 25-hydroxycholecalciferol) resin as the toxic substance in the solvent absolute alcohol. The concentration of cholecalciferol or 25-hydroxycholecalciferol in the solution was about 40% w/v. The formulation was designed to kill stoats.

To confirm the cholecalciferol or 25-hydroxycholecalciferol concentration present and 10 enable accurate exposure calculations when the test solution was applied to the stoats, a sub-sample (approximately 20 ml) of this solution was taken for analysis. Cholecalciferol or 25-hydroxycholecalciferol concentration was determined by taking a sample of liquid which was warmed, mixed and 0.2 g weighed into a volumetric flask. 30 ml of toluene was added, 191143NZ Complete Specification 2009Q602.doc 12 and this solution was sonicated without heating and made up to volume. The solution was diluted with hexane/isopropyl alcohol, filtered and analysed against an internal standard by normal phase liquid chromatography.

Wild-caught adult stoats (three male and three female) were housed in outdoor cages (60 * 5' 150 x 90 cm). Each stoat was provided with a nest box (40 x 33 x 15 cm) containing shredded paper as nesting material. Stoats were fed a rotation of dead day-old chicks, chicken pet mince and mutton/beef pet mince in the afternoon and had free access to water. They were acclimatised to captivity for at least 5 months prior to being used in the trial. The stoats were lightly anaesthetised with isoflurane, weighed and then had 1.5 ml of the test 10 solution applied to their skin and fur, using a syringe to deliver a line of solution along the spine from the nape to the base of the tail. This was a 'high exposure' simulation of the manner in which the delivery system would apply a toxic solution to the fur and skin of stoats.

After application of the solution, the stoats were placed into large nest boxes which were 15 lined with paper but without bedding material, to facilitate observation. Dark Perspex lids were kept on the nest boxes to minimise disturbance of stoats by human observers. The stoats were monitored constantly through recovery from anaesthesia and then for the next three hours to record their responses to the solution on their coats and document any behaviour associated with its presence e.g. grooming, rubbing or rolling. After the three hour 20 observation period in the boxes, stoats were returned to their outdoor cages with nesting material, food and water freely available. They were observed at least hourly for the remainder of that day for any signs of poisoning or responses to the solution. After this, observations of each stoat were recorded once daily, noting any signs of illness that are associated with cholecalciferol or 25-hydroxycholecalciferol poisoning, e.g. stop feeding, 25 significant body weight loss. Where any signs of illness occurred, the monitoring was to be increased to twice daily (morning and afternoon).

Feed intakes of the stoats were visually estimated for at least three days prior to dosing, i.e. ration all eaten, half eaten, quarter eaten, none eaten. Food intake was similarly noted daily after dosing through until death or 21 days (whichever occurred first). Stoats were weighed 30 at least once a week after dosing, through to death or 21 days (which ever occurred first). Any stoats that died during the 21 day observation were to be retained for necropsy. Typical 191143NZ Complete Specification 20090602.doc 13 signs of poisoning (inappetance combined with significant body weight loss) were used as an alternative endpoint for the trial, with a weight loss greater than 25% of bodyweight at dosing triggering euthanasia. Stoats that survived the 21 day observation were to be euthanized.

Results The test solution was analysed as containing 30% cholecalciferol or 25-hydroxycholecalciferol with an uncertainty of ± 5%. This was lower than the nominal 40% cholecalciferol or 25-hydroxycholecalciferol in the solution indicated by the analysis carried out the day after the solution was prepared. The assay was carried out on a sub-sample of 10 solution taken 7 days after preparation, and also following the extraction of 6 x 1.5 ml aliquots of the solution for application to stoats.

During recovery from anaesthesia, 5 of the 6 stoats tried to shake the solution from their backs but otherwise showed normal behaviours and movements for the next three hours. None were observed trying to groom or rub the solution off, one was observed scratching the 15 treated area with a hind foot. The morning after the stoats were returned to the outdoor cages, vomitus was present in three of the 6 cages (female 413, male 391 and male 415). All six stoats showed inappetance from the first night after dosing, generally eating less than half of rations each day. This 'stop feed' effect was considered a typical sign of cholecalciferol or 25-hydroxycholecalciferol poisoning, which was reflected in mortality in three stoats and 20 large losses in bodyweight of the remaining stoats over the following 12 days (see Table 2).

Using the 30% cholecalciferol or 25-hydroxycholecalciferol concentration assayed as a conservative estimate, 1.5 ml of the solution contained 450 mg of cholecalciferol or 25-hydroxycholecalciferol. Table 2 shows the estimated dermal exposures of cholecalciferol or 25-hydroxycholecalciferol delivered to each the six stoats, according to their bodyweight at 25 dosing. The three female stoats died at 4, 11 and 12 days after dosing respectively. The remaining three males were euthanized 12 days after dosing, having triggering the weight loss endpoint - it is highly likely these stoats would have died within the 21 day observation period planned. Necropsy of the stoats that died revealed no gross evidence of calcification/mineralisation or lung damage that has been associated with cholecalciferol or 30 25-hydroxycholecalciferol poisoning in brush tail possums. At death, the site of the solution application was evident from extensive matting of fur. 191143NZ Complete Specification 20090602.doc 14 Table 2. Estimated dermal exposure of cholecalciferol or 25-hydroxycholecalciferol in six stoats, fate, and changes in bodyweight over the 12 days after dosing.

Stoat ID Estimated cholecalciferol or 25-hydroxycholecalciferol exposure (mg/kg) Fate (days after dosing) Weight at dosing (g) Weight at day 7 (g) Weight at day 12 (g) % weight loss at death Male 415 1178 euthanized (12) 382 296 254 36 Male 419 1253 euthanized (12) 359 277 253 37 Male 391 1914 euthanized (12) 235 188 166 Female 414 2205 died (4) 204 na Na 13 Female 407 2331 died (12) 193 151 112 46 Female 413 2486 died (11) 181 120 Na 44 The estimated dermal exposure delivered to the stoats ranged from 1178-2486 mg/kg, with variability largely due to the differences in starting bodyweights between males and females, the latter receiving relatively higher exposures due to their smaller size. The higher cholecalciferol or 25-hydroxycholecalciferol exposures received by female stoats, and their subsequent mortality from poisoning within 12 days (in contrast to euthanasia at the weight 10 loss endpoint in all three males) is suggestive of a dose-dependent time to death. However, all stoats displayed similar timing in onset of signs of poisoning (inappetance) and a similar progression of weight loss. The male stoats that were euthanized were highly likely to have eventually died of poisoning, so the lower end of exposure range probably represents an effective lethal dermal exposure for stoats to this solution (i.e. in excess of or equal to a 15 dermal LD99 value).

The results of this trial suggest that an effective lethal dose of cholecalciferol or 25-hydroxycholecalciferol can be dermally delivered to stoats in the test solution.

It has since been found that the formulations are more effective when delivered in a concentrated amount to a single small area of an animal, rather than a large area. For 20 example, it is preferable for the formulation to be delivered in a circle or spot on the back of 191143NZ Complete Specification 20090602.doc the animal, rather than as a strip or line along the back of the animal. The spot may be about 2 cm in size for example. Accordingly, the automatic dispensing device should be designed where possible to deliver the formulation in this manner. This can be accomplished by narrowing the spray means of the sensor activated dispensing apparatus, for example by 5 passing it through a fine 1 mm diameter pipe, resulting in the release of the toxin to a restricted area of the target species.

The solvent or dermal penetrant used in the formulation should be chosen for its ability to enhance dermal absorption, bioavailability and subsequent toxicity of cholecalciferol or 25-hydroxycholecalciferol. If lower dermal exposures are found to be lethal to stoats, this 10 would entail considerably smaller delivery volumes than the 1.5 ml used in this trial and less cholecalciferol or 25-hydroxycholecalciferol, which would reduce unit costs.

Stability of Formulation Within the stated analysis uncertainty of ± 5%, it is likely there was a slight decline of the cholecalciferol or 25-hydroxycholecalciferol concentration in the test solution during the 7 15 days from preparation to application. This may mostly have occurred on exposure to air when aliquots of solution were drawn from the container for application to the stoats. Cholecalciferol or 25-hydroxycholecalciferol preparations are sensitive to light and exposure to air, with oxidation and inactivation occurring over a few days in the presence of moist air. However, it is noted that even at a 30% concentration, the solution contained sufficient 20 cholecalciferol or 25-hydroxycholecalciferol to deliver an effective lethal dose to all six stoats in 1.5 ml.

Advantages a) The formulation can be administered in lethal doses to the targeted animal pest without having to rely on the targeted pest consuming or continuing to consume an edible poison or bait. b) Reduces the need of dispersing poisoned bait within the environment, and the need for inefficient old style clamping traps. c) Advantages of administering the formulations by way of spray apparatus attached to tunnels or pipes means there is low risk to non-target animals such as dogs, cats, !9i 143NZ Complete Specification 20090602.doc 16 stock and humans as they cannot access the tunnels or pipes. d) Cost effective as only a very small amount of formulation is needed per animal. e) If cholecalciferol or 25-hydroxycholecalciferol is used as the only toxic substance, the advantages include the reduced risk of secondary poisoning to predators and scavengers, low environmental persistence and a degree of selective toxicity to some vertebrate pest species, as well as its low toxicity to bird life and humans.

Variations Although the trials and examples described herein are directed to use on rats, stoats, ferrets and weasels, it will be appreciated by those skilled in the art that the teachings of the present 10 invention can be applied to other mammals, vertebrates, reptiles, and amphibians that are pests which destroy native or endangered flora and fauna. For example, the formulations of the present invention may be applied to cane toads, brown tree snakes, other snakes, frogs, mongoose, foxes and other animal pests.

It will of course be realised that while the foregoing has been given by way of illustrative 15 example of this invention, all such and other modifications and variations thereto as would be apparent to persons skilled in the art are deemed to fall within the broad scope and ambit of this invention as is hereinbefore described.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and 20 unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein; 191143NZ Complete Specification 20090602.doc

Claims (30)

17 this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country. 191143NZ Complete Specification 20090602.doc 18 Claims:

1. A method of killing a non-human animal pest comprising topically administering to the animal pest a topical pesticide formulation comprising an effective amount of cholecalciferol or 25-hydroxycholecalciferol and at least one carrier which is capable of delivering the cholecalciferol or 25-hydroxycholecalciferol transdermally to cause the death of an animal pest.

2. A method of killing a non-human animal pest as claimed in claim 1, wherein the topical pesticide formulation comprises 4 to 50% w/v of cholecalciferol or 25-hydroxycholecalciferol.

3. A method of killing a non-human animal pest as claimed in claim 2, wherein the cholecalciferol or 25-hydroxycholecalciferol is present in an amount of between about 10 to 40% w/v.

4. A method of killing a non-human animal pest as claimed in claim 3, wherein the cholecalciferol or 25-hydroxycholecalciferol is present in an amount of between about 30 to 40% w/v.

5. A method as claimed in any one of claims 1 to 4, wherein the formulation is topically administered to the animal by way of an automatic dispensing apparatus including at least one sensing means to detect when the animal is present.

6. A method as claimed in any one of claims 1 to 5, wherein the formulation is sprayed on to the animal.

7. A method as claimed in claim 6, wherein said automatic dispensing apparatus has means forming at least one partially enclosed space into which the animal can enter or pass through, and at least one dispenser containing a formulation comprising an effective amount of cholecalciferol or 25-hydroxycholecalciferol and at least one carrier which is capable of delivering the cholecalciferol or 25-hydroxycholecalciferol transdermally to the animal, said automatic dispensing apparatus being operatively connected to the enclosed space, said enclosed space including at least one sensing means therein to detect when an animal is present in or 191143NZ Complete Specification 20090602.doc 19 near the space, wherein said sensing means activates the automatic dispensing apparatus to spray a predetermined quantity of the cholecalciferol or 25-hydroxycholecalciferol formulation topically to the animal.

8. A method as claimed in claim 7, wherein the cholecalciferol or 25-hydroxycholecalciferol formulation is sprayed onto the animal from a pressurised aerosol container.

9. A method as claimed in claim 7 or 8, wherein the predetermined quantity of the cholecalciferol or 25-hydroxycholecalciferol formulation is sprayed onto a small area of the animal.

10. A method as claimed in claim 9, wherein the small area of the animal is a spot of about 2cm in diameter.

11. A method as claimed in any one of claims 1 to 10, wherein the formulation is administered to the animal in an amount of between about 0.5 - 2.0 ml.

12. A method as claimed in any one of claims 11, wherein the animal pest is a mouse, rat, stoat, weasel, ferret, rabbit or hedgehog.

13. A method of killing a non-human animal pest as claimed in any one of claims 1 to 12, substantially as herein described with reference to the examples.

14. An apparatus for delivering a topical pesticide formulation to an animal pest, said apparatus comprising means forming at least one partially enclosed space into which the animal can enter or pass through, and at least one automatic dispensing apparatus having a spray means, and a container containing a formulation comprising an effective amount of cholecalciferol or 25-hydroxycholecalciferol in a solution which is capable of delivering the cholecalciferol or 25-hydroxycholecalciferol transdermally to the animal, said automatic dispensing apparatus being operatively connected to the enclosed space, said enclosed space including at least one sensing means therein to detect when an animal is present in or near the space, wherein said sensing means activates the automatic dispensing apparatus to spray a predetermined quantity of the cholecalciferol or 25-hydroxychorecalciferol formulation topically to the animal. 191143NZ Complete Specification 20090602.doc 20

15. An apparatus as claimed in claim 14, wherein the spray means and the container comprise a pressurised aerosol container.

16. An apparatus as claimed in claim 14 or 15, wherein the predetermined quantity of cholecalciferol or 25-hydroxycholecalciferol formulation is between about 0.5 - 2.0 ml.

17. An apparatus asf claimed in any one of claims 14 to 16, wherein the formulation in the container comprises about 4 to 40% w/v of cholecalciferol or 25-hydroxycholecalciferol.

18. An apparatus as claimed in any one of claims 15 to 16, wherein the formulation in the container comprises about 10 to 40% w/v of cholecalciferol or 25-hydroxycholecalciferol.

19. An apparatus as claimed in any one of claims 14 to 18, wherein the container contains a formulation comprising from 30 to 40% w/v of cholecalciferol or 25-hydroxycholecalciferol in solution.

20. An apparatus as claimed in any one of claims 14 to 19, substantially as herein described with reference to the examples.

21. A topical pesticidal formulation comprising about 4 - 50% w/v of cholecalciferol or 25-hydroxycholecalciferol and at least one carrier which is capable of delivering the cholecalciferol or 25-hydroxycholecalciferol transdermally to cause the death of an animal pest.

22. A topical pesticidal formulation as claimed in claim 21, wherein the cholecalciferol or 25-hydroxycholecalciferol is present in an amount of between about 10 - 40% w/v.

23. A topical pesticidal formulation as claimed in claim 21, wherein the cholecalciferol or 25-hydroxycholecalciferol is present in an amount of between about 30 - 40% w/v.

24. A topical pesticidal formulation as claimed in any one claims 21 to 23, wherein the carrier or each carrier is selected from the group comprising absolute alcohol, 19U43NZ Complete Specification 20090602.doc 21 ethanol, isopropanol, methanol, benzyl alcohol, glycol ethers, glycerol formal, polyethylene glycols, liquid polyoxyethylene glycols, propylene glycol, pyrrolidones such as N-methylpyrrolidone and 2-pyrrolidone, acetone, acetonitrile, butyl diglycol, dimethyl sulfoxides, dimethylacetamide, dimethylformamide, monomethylacetamide, diethyl phthalate, or any combination thereof. \

25. A topical pesticidal formulation as claimed in claim 24, wherein the carrier or one of the carriers is absolute alcohol.

26. A topical pesticidal formulation as claimed in any one of claims 21 to 25, wherein the formulation is in the form of a foam, gel, or liquid spray.

27. A topical pesticidal formulation as claimed in any one of claims 21 to 26, wherein the formulation further includes calcium.

28. A topical pesticidal formulation as claimed in any one of claims 21 to 27, wherein the formulation is a liquid adapted to be dispensed as an aerosol spray.

29. A topical pesticidal formulation as claimed in any one of claims 21 to 28, wherein the formulation is contained within a pressurised aerosol container.

30. A topical pesticide formulation as claimed in any one of claim 21 to 29, substantially as herein described with reference to the examples. PIPERS Attorneys for the Applicant WARREN ROY AGNEW 191143NZ Complete Specification 20090602.doc