Community Research and Development Information Service - CORDIS

FP7

PIEDPIPER Report Summary

Project ID: 286852
Funded under: FP7-SME
Country: United Kingdom

Final Report Summary - PIEDPIPER (Piedpiper)

Executive Summary:
The project PiedPiper was devised by Steve Goode after he had seen an ever increasing number of news articles expressing increasing concern over the rise of rodents in general and rats in particular to the current toxins.
This lead to some research into the problem and it was soon established that the cause of the problem was not the products per se but the actual method of use that had been used. These Warfarin and Second Generation Anticoagulants were developed initially with the warfarins in the 1950’s then the Second Generation Anticoagulants in the 1960’s onwards and at the time were hailed as the ultimate in pest control. The problem with these toxins however started to be documented over the years, Resistance to the toxins became evident and problematical. Today you cannot pick up a major newspaper from anywhere in the world without it having an article in about resistant rodents. So what is the problem and why did it all go so wrong for these compounds?
In high insight the problem becomes all too clear. The route of administration for these toxins was oral in food compounds (prepared bait based on linseed or wheat. The bottom line is that the rats did not ingest enough in a single feed to kill the rodent. The system employed is called multi-feeding and the result was that “partially” poisoned rats were able to survive and breed. In surviving they also built up antibodies which were passed on the next generation in the mother’s milk. Generations of this ratcheting up the antibody levels has resulted in resistance of these rats. This situation has to stop somewhere but the industry is proposing using higher and higher dose levels but it does not provide a solution.
We had to look for an alternative system that would be effective and preferably allow us to reset the clock by enabling us to kill all of these warfarin / SGAC resistant rats whilst at the same time removing the risk of repeating the resistance problem with another compound. A product that could kill in one application was essential and be able to minimize the suffering to the target animals. We needed an innovation could we develop a technology that delivered a rat / mouse lethal dose onto the fur? Then it needed to be absorbed through the skin and into the bloodstream to deliver a fatal dose. This is where our One Shot = One Kill – Each Time - Every Time slogan came from and became the cornerstone of our technology. We also had other criteria – user friendly, environmentally friendly and we wanted to stop the secondary kills, specifically cats, dogs and birds of prey (This is where a dead / dying poisoned rat is eaten by another animal or bird.). The system also needed to be safe to humans and be a multi shot approach to keep user costs down. By keeping costs down I mean that the canister should hold a number of doses so unlike standard snap traps and bait stations they did not need frequent attention. The other big issue was how much toxin was being placed in the environment by these current toxins – too much is the straight answer. Our system was devised to only deliver a toxic dose – the majority of which would be metabolized and cause its death. We initially thought we would be putting 5 ml of toxin into the environment which was just about on the edge of the current technology. In the end we have reduced that to 1 ml of toxin per rat (mice 0.25 ml). This putting poison into the environment is called – environmental toxic load (ETL). In essence this is one of the biggest developments form our project = ETL. One pest control expert worked out that he had used 100,000 + tons. One pest control expert worked out that he had used 100,000 + tons of toxins in his professional work and with our system that would be reduced to around 100 tons. Another and important issue was to avoid pollution of areas such as outdoor areas and sewers where a high proportion of rat poisons are used. Our toxin is virtually insoluble in water compared to the currently registered products which can easily destroy entire aquatic ecosystems. PiedPiper was challenging – not only the toxicology but the PCD (Pest Control Device hardware) and the work to attract rats to the trap but due to the hard work of the Piedpiper Consortium we have delivered a revolutionary system.

Project Context and Objectives:
Historical Overview

In 1948 Warfarin was introduced as a poison for rats, it quickly gained popularity as it was highly effective and easy to use. There was however an enormous mistake made with Warfarin and also the second generation anticoagulants such as Brodifenicum which was not realized at the time but over the intervening 50 – 60 years has become apparent. The products were used in a multi-feed adlib format and over the years rats gradually built up a tolerance to these compounds. To be fair to the manufacturers and pest controllers of the time they had no inkling of the magnitude of the problem that was being created nor in reality did they have guaranteed controlled delivery methods. The amount of poison needed to kill rats and mice has increased as they have become resistant to Warfarin resulting in “super rats” as the press have dubbed them. The scientific fact is that the problem is getting worse year on year and will continue to do so whilst there is a fundamentally flawed toxin usage policy for rodent control.

The pest control industry and the manufacturers of these products have lobbied strongly to get the EU to reverse the ban. It is not going to happen – at least it should not happen – if Warfarin continues to be used rats will become effectively immune to this type of poisoning. In addition Warfarin has a number of serious drawbacks for its use as a rodenticide. It is readily soluble in water and highly toxic to aquatic ecosystems. Secondary kills (poisoning or death of a non-target species) occur when dead or severely poisoned rats and mice are eaten by cats / dogs or birds of prey. Unfortunately birds tend to die due in part to their size relative to their prey and also the fact they are wild animals and not easily treatable. The cats and dogs which ingest warfarin toxin laden rats can, in a percentage of cases can be saved by veterinary attention. There is a cost to this – and surprisingly if you live in the countryside it does not appear to be that unusual an event. The bills for veterinary attention are generally in the region of 1- 2000 Euros.

Another issue which became apparent during the PiedPiper project is that the current Pest Control methods result in a high wastage of the toxins. This was drawn to the consortium’s attention by the owner of a New Zealand Pest Control business. There the regulatory controls are beginning to change with an emphasis on the environment and how much toxin is actually being put into the environment i.e. the Environmental Toxic Load. A Pest Controller reported to BIOT that he had calculated that in his time in pest control he had put some 115,000 tons of toxins into the environment and a lot of this has been wasted – some gets broken down over time, washed or blown away and in the best case scenario picked up and disposed of professionally. Of that mass of product in the environment most of it is available to poison non-target species.

Current Position

The EU has been very proactive in tackling the problem and has taken the brave, logical and correct decision to enforce a ban on the use for pest control of Warfarin based products from May 2014. The PiedPiper project came about as a direct response to this to provide a practical, safe replacement to what have become outdated products. The PiedPiper technology uses as the toxin a naturally occurring compound -Vitamin D3 delivered at a dose level which is toxic to rats but presents no danger to other species. Rodents are particularly sensitive to Vitamin D3. Once the rat has been treated it dies very quickly with little suffering. The time from onset of symptoms to death is normally less than 10 hours compared to up to 6 weeks for current products. One dose of our poison results in death, and because of the method of application the rat population will never develop a resistance to Vitamin D3. A further benefit is that Vitamin D3 is metabolized up to and after death. Thus the problem of secondary kills is solved as the body of the dead rat – if consumed by a cat, dog or bird of prey contains only a therapeutic dose of Vitamin D3. It also needs to be pointed out that nocturnal animals are very sensitive to vitamin D3 whilst humans, cats, dogs and birds of prey are not at all sensitive. Using the PiedPiper technology could result in a significant reduction in the amount of toxin in the environment. The New Zealand Pest Controller has calculated that if he had used the PiedPiper system the quantity of toxin he had put into the environment would have dropped to a few tons. This has to be a major step forward in pest control technology.

Warfarins are in their final stages on the market but Second Generation Anti-Coagulants will still be available on the EU and world markets. They are going to be reviewed but there is a vociferous industry lobby to have them retained and unbelievably change the regulations to allow more widespread use of the products and worse still enable higher concentrations to be used. We are looking at moving from the super rats to a whole new level of tolerance to these products if we proceed down this path. From their perspective the industry has a vested interest in preserving their chemical production, pest control operations and of course profits – i.e. the status quo. It would be fair to say that pest control regulations have not been top of any regulators agenda until recently. The pest control industry has had a good run – 50 – 60 years using essentially the same compounds and little or no R and D costs to develop new products. Given the overwhelming evidence of the situation now with regards to efficacy / tolerance against these products it is time to move on and use a new system. To continue to allow the industry to use these products in higher concentrations and in less secure locations is just asking for environmental problems. What it needs is for everyone to step back and take the emotion out of the situation.

The purpose of the PiedPiper project and the grant was to produce an independent solution that addressed all of the problems of the current toxins and would provide a viable humane effective system to move forward with. The industry should be embracing the PiedPiper technology as a step change to a new safer, more efficient approach to rodent control. This document is the final document for the PiedPiper FP7 Grant (R4SME 286852) and marks the beginning of the journey to market for this new technology. The PiedPiper acknowledge and thank the EU REA for our FP7 Grant which was specifically aimed at assisting aspiring innovative SME’s by providing funds for them to spend on Universities and other research facilities to carry out work that they could not either do themselves or afford to do. The amount of public funds (1.2 M Euro) on top of smaller grants from MAS (Manufacturing Advisory Service) and AWM (Advantage West Midlands) have been a great assistance in getting this project to the state it is today.. In addition PiedPiper has also received Mentoring support from Connect West Midlands, Business Link and other UK organisations which offer consultancy support to innovators and assist high growth potential companies. The response PiedPiper has had from these organisations and potential investors has been overwhelmingly positive and our discussions with industry pest controllers and the public has also been highly positive.
The research activities for the project officially finished on 30th November 2013 but all of the RTD’s and SME’s have continued to work on finalizing their individual work tasks. The developments and findings will be reported in peer reviewed papers by the RTD’s and work is ongoing to protect the IP. In the meantime there is a BBC News article on the PiedPiper project – just follow this link : http://www.bbc.co.uk/news/science-environment-25573771
PiedPiper had only been operating for 9 months when we did our first clinical trials. So positive were the results that I wrote a second grant application for an FP7 demonstration grant – called a DA or “Demonstrator Activity”. This was written based on the first 9 months research and was filed a matter of hours before our 9 month EU REA review. In March 2013 we heard that PiedPiper had been awarded the Demonstrator Activity Grant under FP7 funding which will support pre-commercialisation activities.
The project will need funding to get through the regulatory process and onto the EU market to replace the current range of products. Initial outline discussions with a number of English, Welsh, Australian, Middle Eastern and US groups have been very positive. We have in parallel discussions with partner companies ongoing. All of these groups find our approach innovative – novel, practical and overall much needed.

So what is PiedPiper and what has it achieved.

We started with a blank sheet of paper and after reviewing toxicology papers and industry practices it was clear a new approach had to be developed. We opted to look at Cholecalciferol (Vitamin D3) as the toxin – it had already been used in small quantities in the past as a rat poison the delivery of the toxin had to be different from the current practice of multi-feeding pellets or blocks. The next challenge was how to get toxin into the rat effectively. The answer was to develop an aerosol system that carried the toxin and a solvent to carry the toxin through the skin into the rats system – i.e. bloodstream. This was the biggest single part of the research project and was carried out by Aston University under the appropriate Home Office Regulatory procedures and certification. The bar was set high in terms of requirements – the process had to be humane, causing minimal stress and a short time to death as possible. This has been achieved – rats show no signs of poisoning up to 48 hours then after a further 10 hours nearly all have died. The system is 100% effective – with the standard dose it works every time. One shot = one kill, each time – every time.
This system has a number of significant advantages – the primary one being that there is no chance of rats becoming immune / tolerant to our dosage. The dose rate is very low – and will not kill other animals such as cats, dogs or birds of prey – either directly or via secondary kills (i.e. consumption of dead carcasses). What we have produced is a unique technology that is patented and has additional patents applied for. We believe we have created a product that is environmentally friendly and has the lowest environmental impact and lowest environmental toxic load of all rodenticide products. By nature of it being aerosol based it is multi shot and this allows a significantly longer period between maintenance visits. We have plans to add networking as an option to allow more specific targeting – further cutting down on labour and travel costs and hence lower costs per kill. Due to the low solubility of Vitamin D3 it is unlikely to be a pollution risk like the current very soluble products. Our product is unlikely to get into the wider environment. Primarily because it is sprayed directly on to the back of the rat within our “run through” device (PCD) and is then rapidly absorbed through the skin. Secondly the toxin is contained within an aerosol canister (securely locked to the PCD) and is not free in the environment to be washed away by rain or drainage water. The final part of the project was to screen rodent urine and other products to isolate attractant compounds to add to the pest control device which means that food bait will no longer be necessary..

Project Results:
Science and Technology - Stakeholder targeting
- This was an early deliverable in the first reporting period but as project coordinator I have spent a lot of time and effort to talk to all sections of the population who may have an interest in pest control and rats in particular – The Website has been updated and eventually split into two to address the various diverse markets that affect our stake holders. Being a coordinator of an FP7 project is not the easiest job in the world it requires dedication and the ability to think in 3D. We were unlucky in our Project in that one of our consortium members had to be removed from the PiedPiper consortium as they unfortunately were placed in Liquidation in March 2013. This had a knock on effect through the consortium as we had to discuss whether we sought to include a replacement for them or if the remaining work could be carried on in the consortium. In conjunction with discussions with the project officer for PiedPiper we decided that it was best to take the work from Helios and place it with Cellvax. The fortunate issue for the group was that Helios delivered a major part of their work at a consortium meeting immediately prior to their liquidation. The remaining work was a statistical analysis of the rat kill trials. The solution agreed with the EU REA in the DOW (description of Work) was that Cellvax or Aston could have undertaken the work. We decided to accept Cellvax’s offer to carry out this work and have the DOW modified to reflect these changes.
The consortium members have all been very interactive and communicated freely on the various issues and problems that have inevitably arisen during the project. The best example of this stakeholder input being important is in production of the prototype. The collaboration exemplifies the EU concept of FP7. Aston had a significant input in terms of toxin delivery relating to the specification of the toxin and its effects on the materials. Burgos had their input relating to the location and method of release of the attractant molecules. Helios when they were still members had significant input in terms of how these molecules would perform and persist in the environment. Cellvax contributed directly to the specification of the prototype with their experience of 10 years of running rat test facilities at Cellvax and involvement in numerous rat clinical trials prior to this. They commented on the behavioural aspects of rats and their sensitivity to changes in their environment and the smell / appearance/ colour of these items. IRIS had the input from their environmental engineering position as the RTD that was tasked with the building of the prototype. Biotronics has the original concept and drawing s which we had produced by Wolverhampton University after submitting our original specification then working with the engineer who was tasked to produce the drawings and proof of concept model for the AWM (Advantage West Midlands – our regional development agency). The biggest technical input for the aerosol task was that of Thomas Huurman of Summit Europe who specializes in aerosol valve design and supply. Their knowledge of aerosol valve technology was essential to this task and was invaluable. Thomas identified a number of prospective problems that we essential to overcome for the aerosol technology to work. The first issue was the aerosol valve seating gasket – which is normally a rubber compound – that swells and goes soft with the toxin formulation – this was resolved by testing of other compounds – neoprene being a suitable material to use. The valve crimp body was also a potential problem and has been resolved with fill / extended environmental tests using other plating materials.
In conclusion as a group of SME’s and RTD’s formed into a consortium I doubt that you could get better communication and interaction. The stakeholders have remained focused on the tasks required to be addressed and the problems resolved by the coordinator. The results achieved by the PiedPiper consortium are a testament to the effectiveness of communications and suitability of skill sets for the project.
WP 3
WP3 Validation of a Toxin
3.2.1 Project objectives for the period
Overview of the project objectives for the reporting period
• Determination of the acute oral toxicity of vitamin D in Rats.
• Physical characterisation of the proposed active materials
• In-vitro model development
• Formulation development
• Penetration enhancement
• Life cycle Analysis
No recommendations from the previous reviews were considered or made.
Progress towards objectives and details of tasks
Clearly significant results are highlighted.
O3.1 To determine the toxicity of the vitamin D or analogue to be cause rodent death.
During the previous initial research period; Vitamin D3 had continued to be a potential for use in all aspects of the formulation both its in-vitro properties and physical compatibility properties were favourable. Existing data was previously documented for this compound for oral toxicity levels and to a very limited level some data speculated toxicity through a contact route. Having presented the literature review which was agreed would have delayed publication due to confidentiality the priority was to move towards in-vivo work with priority formulations. Rodent death through oral delivery and the specific poison were confirmed as useful for the research aims. O3.1 was concluded complete.

O3.2 To deliver the toxin to the systemic circulation of a rodent in a way that minimises the risk to human or other secondary organisms.
Properties for transdermal delivery of concern to the project included poor aqueous solubility as well as accelerated degradation. The in-vitro transdermal work confirmed systemic levels similar to oral toxicity may be possible using a completely organic solvent based system but that would have degradation and freezing point problems. In these systems we showed that toxin concentrations could be controlled to provide a suitable volume deliverable through the proposed spray. It was noted that we quickly extended the large solubility changes to provide a lower volume for delivery to the rat/pest. This allowed much more precise control of the toxin and limited harm greatly to humans. The formulation although toxic to humans had a dosed level expected to be significantly less than those required to have clinical affect. This including been significantly below the single reported case of high level human intoxication with the crystalline API which although been an extreme case of overdose had only resulted in a calcium imbalance corrected with steroid treatments by the medical team. This reassured the RTD team that human danger from this toxin would allow a safe and reasonable balance of hazards both for working in the laboratory but inherited into the final formulation.
In-vivo work was conducted based on OECD guidelines under full ethically approved conditions within Europe. Initial studies confirmed levels of significant toxicity in small group numbers [Figure 1] with a single priority formulation moving forward to testing to the level of significance required by the European biocide directive. In all cases the mortality from this formulation was 100% in excess of that required for inclusion as a biocide agent [Figure 2].

O3.3 To control the solution or suspension properties to aid high levels of transdermal and oral delivery.
The compressed butane delivery systems proposed would heavily restrict or demand huge engineering changes to the final device if a suspension was to be used. A solution state formulation therefore remained a priority. Viscosity and wetting were asset in a simple excised rat tissue in-vitro model. Formulation selection included a balance of agents shown to provide broad working temperature ranges while maintaining or not further increasing freezing points [Figure 4] to provide a formulation capable of deployment in harsh (subzero) environments. Chemical skin disruptants were selected and refined with similar priorities through in-vitro testing.
O3.4 To develop a model for further product optimisation and use in the quality assurance process for final routine formulation batch release.
Both the transdermal in-vitro model and HPLC method were available in the first reporting period. Using the in-vivo data for the vitamin D3 levels there appears to be a usable correlation that will allow formulation modifications in the future. The initial in-vitro model was further simplified to use a more reproducible single membrane system making the method more usable in a high throughput environment.
The HPLC methodology has been refined to reduce retention time while including steps to provide internal standards as well as allow separation of some of the apparent impurities on storage. This has benefitted accelerated storage testing of the formulations and review [Figure 3].
O3.4 Concluded complete – with open ended improvements to reflect continuous development.

Reasons for deviations from Annex I and their impact on other tasks as well as on available resources and planning
Rework of the UK animal licence was altered to include elements of WK 4 when it was realised that the method of analysis of pheromones in-vitro would be unsuitable. This delayed submission and return but would allow performance analysis of a limited number of specific molecules. This pushed final in-vivo assessment of formulations in WP3 but would allow the potential for significant conclusions to be drawn for WP4. This was done with full knowledge and supervision of the co-ordinator.

A statement on the use of resources, in particular highlighting and explaining deviations between actual and planned person-months per work package and per beneficiary in Annex 1 (Description of Work);
O3.1 provided reduced levels of risk in comparison to the potential for work with less well known analogues of the proposed poisons. It was frustrating this funding could not be better internally allocated to benefit wider elements of WP3 or released wider.
Vermin Attraction WP4
Activity type: RTD – Research.
Objectives:
O4.1 Identification of rodent pheromones and attractants.
O4.2 Design synthesis of rodent pheromones
O4.3 Synthesize unnatural pheromone analogues
O4.4 Design a convenient cost-effective synthesis for lead compound scale up production
Description of work:
T4.1 Identification of rodent pheromones
T4.2 Synthesis of pheromones
T4.3 Preparation of unnatural analogs
T4.4 Identification of lead compound and design of synthetic route
Deliverables that have been files within this project.
D4.1 Literature Review- Month 3
D4.2 Identification and synthesis pheromones Month 12
D4.3 Design of scalable synthesis of lead pheromone Month 15
Pheromones can be classified as a class of “semiochemicals”, substances carrying a chemical message among animals. This term was first introduced in 1959 by the entomologists Peter Karlson and Martin Lüscher to identify specific biologically active substances “which are secreted to the outside by an individual and received by a second individual of the same species, in which they release a specific reaction, for example, a definite behaviour or a developmental process”. Since then, pheromones have attracted an enormous interest from the scientific community and examples of this type of compounds have been identified from species in every animal phylum. The use of pheromones as triggers of behaviour and pest control is nowadays well established for insects. On the other hand, similar applications in the case of mammals and other animals are largely unexplored.
Rodents’ ecology is largely triggered by pheromonal signals, which have been investigated for a long time, and therefore, these species represent the standard animal model to study interspecies communication. These studies usually involves laboratory species, among which rodents and especially mice are the most employed.
All body secretions are potential routes for pheromonal communication and many mammals have developed specialized scent glands that they use to deposit scent marks around their environment. In this regard, urine is especially important in controlling rodent social interactions. A literature review was compiled in deliverable D4.1.
In order to address the objective O4.1 “Identification of rodent pheromones”, task T4.1 “Identification of rodent pheromones” was initiated. We decided to analyze rodent urine samples using solid phase micro-extraction (SPME) coupled to gas chromatography/mass spectrometry.25 Solid phase micro-extraction was developed to address the need to facilitate rapid sample preparation. In this technique a small amount of extracting phase dispersed on a solid support, is exposed to the sample for a well defined period of time. This technique presents several advantages such as small sample volume, easy coupling with GC and the potential to use different fibres (CAR/PDMS). Moreover SPME can be used in a complex multiphase system and speciate different forms of analytes in a sample. It is a solvent free, economical, and versatile technique with low detection limits. There are different parameters which can be adjusted in the SPME procedure: Extraction time, temperature, agitation/stirring and pH. All of them were trialled in order to find the optimum extraction conditions.

Figure 1. SPME of a rodent urine sample (left) and Gas chromatography/mass spectrometry (GC/MS) equipment (right).
After the adsorption process on to the solid coating of a SPME fibre, a desorption step is needed to introduce the sample in the GC/MS for the analysis. In order to achieve an effective desorption the needle is introduce in to the GC and fiber was exposed for a few minutes to a carrier inert gas (He). Finally the carrier gas was introduced in the chromatographic column for the analysis. With the mass spectrometry detector system the identification of individual compounds is possible. Compounds were assigned an identified using the NIST mass spectral search program.

Figure 2. Extraction and Desorption procedure in SPME
Figure 2. Gas chromatography/mass spectrometry (GC/MS) equipment.
As a result of these analysis, a total of 31 compounds were found and identified in rodent urine samples:
Based on these results, a library of compounds was selected in order to be tested as pheromone attractants. The selection included compounds such as ketones, aldehydes and alcohols with both low bowling points and low molecular weight, and structurally related to compounds identified as pheromones with different functions on several species. The selection of compounds was based on the following criteria:
• All of them belong to families of compounds previously identified as pheromone/attractants in the literature either for rodents or other species.
• Because of their nature a wide range of boiling points/volatilities was covered. We aimed to use compounds as attractants and we anticipated the persistence and volatility balance as an important parameter to be considered.
• Being a selection of pheromone candidates we aimed to select commercially available compounds as first candidates. Therefore, safety data and related information were available for all these compounds. In this regard all regulatory issues would be easier.

Figure 3. Selected candidates as rodent pheromone/attractants identified in natural samples.
A study of commercial suppliers, prices, purities and safety of all compounds was performed by Helios Biosciences and presented in the PiedPiper meeting at IRIS in February 2013. Additionally, LASSO scores for all these compounds were determined. LASSO is a searching tool that uses LASSO (Ligand Activity in Surface Similarity Order) descriptors to find molecules with diverse chemical scaffolds but similar surface properties. It is based on the idea that ligands must have surface properties compatible with the target site in order to bind; LASSO uses a descriptor of Interacting Surface Point Types (ISPT). The LASSO descriptor provides fuzziness in terms of molecular structure which makes it ideal for scaffold hopping applications. It is expected that pheromones function by binding to specific receptors. LASSO descriptors allow a straightforward comparison of structurally diverse molecules in terms of surface similarity. Moreover once a molecule is identified as an attractant pheromone it allows finding other molecules with similar binding properties. Using Lasso scores similar compounds can be proposed if a second generation of pheromone attractants need to be tested.
In order to reduce the cost of all compounds Synthetic routes were proposed. All this information was included in deliverable D4.3. The proposed synthetic routes were based on starting materials that were commercially available in bulk quantities and inexpensive. Moreover the proposed synthetic route delivered the target compound in high yield. The selected reactions involved just one step or the different reactions required to yield the final product could be carried out without the need for isolating the corresponding intermediate compounds. These reactions were based on published procedures and are not covered by patents and the reactions were environmentally friendly and toxic reagents were avoided as much as possible. This work correspond to O4.2 Design synthesis of rodent pheromones
In the interim review meeting covering the first 9 months of the project, the scientific reviewer suggested to expand our studies to other rodents. Thus mice urine samples were also analyzed. Our goal in this work was to optimise the SPME conditions using an experimental design to carry out the analyses and to establish the best extraction conditions for volatile compounds in mice urine samples. Moreover we wanted to compare mice urine samples of animals of both sexis and in the case of female individuals in the different stages of the reproductive cycle. Applying the optimised extractions conditions (108 µl, 148.6 ºC and 94 minutes) resulted in robust, reproducible results. 36 organic compounds were detected in those samples. Several differences could be observed when comparing male and female mice urine samples. Benzaldehyde was found to be the main compound detected in female urine samples. Depending on the gender specific compounds could be detected in the samples. Thus compounds such as 2-isopropyl-4,5-dihydrothiazole were exclusively detected in male samples whereas ketones like 4-octen-2-one, 3-octen-2-one were detected in female urine samples. Part of this work was recently published as a full paper entitled “Determination of Volatiles in Mouse Urine by Headspace Solid Phase Microextraction and Gas Chromatography-Mass Spectrometry” in the journal Analytical Letters (DOI 10.1080/00032719.2013.853182). This work was also presented at two different chemistry meetings by the PiedPiper team at UBU.

Figure 4. Response Surface applied in the optimisation of HS-SPME extractions in mice urine samples

One of the challenges faced by PiedPiper consortium is how to incorporate the pheromone attractants to the PiedPiper prototype. There are different materials that could be used for this purpose. The goal is to find an appropriate system capable of gradually release the pheromones to the environment. This system should be cheap, non toxic and compatible with different types of pheromones. The pheromones would be spiked on to the material when produced and once the container is exposed to the environment a continuous flow of the pheromones would be released until exhausted.
Positive controls analyses.
In our last meeting at Aston University it was decided to test both peanut butter and rat food pellets as positive controls in the rodent behavior experiments. UBU was decided to determine volatile compounds in both types of samples. We found in the literature such type of analysis for peanut butter. Using the same techniques as those used to study rodent urine samples, volatile compounds from food pellets were determined. The results are described below.
Peanut butter.
Studies regarding composition of peanut butter volatiles were found in the literature. These studies aimed to determine desirable characteristic of peanut butter aroma, their changes upon roasting processes and the characteristics of normal vs. rancid peanut butter.123 94 volatile compounds were identified and characterized by HS-SPME/GC- MS. Among these were pyrazines, pyridines, pyrroles, furans, pyran, aldehydes, alcohols, ketones, acids, alkanes, alkenes, and esters. Most of the nonheterocyclic chemical classes such as aldehydes, ketones, acids and esters were derived from lipid decomposition and among these; aldehydes compounds were considered the main significant components for flavor of vegetable oil. Pyrazines are also considered to be important for the typical roasty/nutty flavor of peanut butter. Rat food pellets analysis.
24 different compounds were identified in our analysis of the pellets (see annex I for details). Among them aldehydes and ketones were the most abundant derivatives. Two pyrazines and other heterocyclic compounds were also identified a link with several alkanes and alkenes. The last two classes of compounds are not considered to impact significantly in the flavour score of food items.
Figure1. Representative TIC chromatograms in SCAN mode of SPME extracts of rat food pellets.
1 Braddock, J. C., Sims, C. A., and O’Keefe, S. F.: Flavorand oxidative stability of roasted high oleic acid peanuts. J. Food Sci., 60, 489–493 (1995). 2 J. R. Vercellotti, A. J. ST. Angelo, M. G. Legendre, G. Sumrell, H. P. Dupuy, and G. J. Rick, JR. Analysis of Trace Volatiles in Food and Beverage Products Involving Removal at a Mild Temperature under Vacuum. Journal of Food Composition and Analysis 1, 239-249 (1988) 3 X. Liu, Q. Jin, Y. Liu, J. Huang, X. Wang, W. Mao, S. Wang. Changes in Volatile Compounds of Peanut Oil during the Roasting Process for Production of Aromatic Roasted Peanut Oil. Journal of Food Science Vol. 76, Nr. 3, c-404-411 (2011)
Conclusions.
Both rat food pellets and peanut butter show a volatile profile sharing characteristic families of compounds such as aldehydes, ketones and pyrazines. The composition of attractant gels delivered to Aston also matched these types of compounds. Thus chemicals such as benzaldehyde, hexanal and some pyrazine derivatives were included as attractant candidates. In this regard our selection of attractants seems appropriate. In any case it is yet to be studied whether a single chemical can trigger the attraction of rodents as the complex mixture corresponding to real food samples do.
Development of Prototype: Pest Control Device.
At the first periodic review IRIS presented the initial prototype that was presented in clear plastic and was our first attempt to improve on the original concept design produced by Biotronics prior to this project proposal being submitted. See Fig a.
Figure a Figure b figure c
The clear Perspex design had some shortcomings – not only structurally i.e. being clear but also the material it had been fabricated from was not resistant to our toxin / solvent formulation. The meeting held in Castelldefels (IRIS) in February 2013 was an opportunity to sit down and have a substantial reappraisal of the entire design. All members of the consortium including Helios contributed significantly to the re-specification of the prototype and over the following two months we had an ongoing dialogue with all members of the consortium. We could have left the clear design as the final prototype but the level of enthusiasm and commitment in the project led us to revisit the specification. The culmination of the feedback from the members resulted in Joe and Andrew collating a revised “wish list” (this effectively re-specified the PCD and took into account results of recent toxicology work at Aston.) which was discussed with IRIS at our MAY meeting at Summit in Almere. At this meeting we were presented with the first of the “Red” PCD units – which were a revised design item for discussion. This is the point at which we finalized the design that was presented at the PiedPiper final appraisal. Meeting at IRIS on 12th December. There have been a number of updates in design, software / interface and construction – minor changes but nevertheless progress towards a pre-market product that we will develop for the PiedPiper DA. This sort of incremental improvement is typical of product development and is always highly dependent on the level of support from the consortium. The biggest single upgrade to the specification has been the use of a red polyethylene sheet that is opaque and inert to exposure to solvents used in the toxin formulation. The reference for this is:
(http://www..arkema-inc.com/literature/pdf/361.pdf).
IRIS also carried out accelerated environmental testing work on the PCD. This has been completed and submitted and D5.4 has been filed as a deliverable.

Potential Impact:
One of the most interesting visits of the year was to Pestex Exhibition – Excell Centre, East London - April 2013– I took two guests who are interested in the PiedPiper pest control market from an investor’s point of view. There were 25 major MBO’s and purchases in 2012. There is evidence that the market is “professionalizing”. The Guests were senior business managers and accountants they were both singularly unimpressed with the level of sophistication displayed at such a premier UK /EU industry event. The accountant involved did a market analysis of the PiedPiper business assuming prices for regulatory approval would not exceed 1.5 – 2.0 M Euros. The cost per kill against Warfarin / Second Generation Anti-Coagulants (assuming that the industry would get it to remain for a further period of time in the EU market) were calculated and his pricing came out at the equivalent of Warfarin. His figures were based on our initial dose (based on US papers which document that as a kill dose). He also confirmed the fact that we would have a lower environmental toxic load as all of our product was used – sprayed directly on the rat – max volume 1ml per dose compared to significant quantities of bait block.
ARC Associates - presentation to prospective investors / partners including a Philippines based business – located in Manila.
Presentation to Dr Huw Edwards who is in the EU REA and is on the FP7 committees (He works in the field of drug delivery and nano-technology) - and works as a scientific technology consultant one of the industry’s premier players. The presentation was given by Andrew Ingham / Steve Goode and after a presentation we had a question and answer session.
–Meeting with a large manufacturing association. I spent a morning with the CEO who is very forthright about business and whether they have the capacity to be successful. The reason for meeting him is he has a Manchester based manufacturing group and has 40 years of so experience in manufacturing, consultancy and mentoring. We went through my background, education, experience and what I have already invented / achieved. We then discussed PiedPiper and the PCD at length as the consultant was looking for a problem that we have not foreseen in the technology or the manufacturing concept. He then analysed the market with me and then we discussed financing the project and what type of investor / partner would be most appropriate / beneficial. It was an interesting exercise which he tells me in the case of most projects shows up flaws in the concept, markets, production etc. He found none in our project or the processes we had undergone to get to the stage we are today. His comments were actually very complimentarybut he did say that finance was not easy in the current economic cycle. I came away from the meeting happy that we have done everything we can as a consortium and as an individual coordinator.
As indicated in the initial proposed PUDF there are the following requirements for knowledge management, dissemination and exploitation. These further divide into:
1) Acquisition of knowledge and the reviewing of the information with regard to which remains confidential and which goes to public domain.
2) Once we have decided to classify items as public domain we then have to decide which medium we can use. (Press release, Twitter, Website, exhibitions (visiting and exhibiting) brochure, IP- patent etc, academic paper or presentation format.) All of these have been used during the course of this project.
3) IP is a very difficult management process – and we have had a substantial number of meetings with patent attorneys during the course of this project. The IP routes we have decided to use are patent applications and also the final preproduction prototype will have a registered design application submitted to secure that IP.
4) Pest control industry – this again is very difficult to manage and we have used NDA’s for the academic end of this spectrum e.g. a university based in the south east and a team based team at a northern university. This is a situation where even though there is an NDA in place we have to be cautious as these teams are sponsored by the “big business” pest control and chemical manufacturing sector. Small pest control businesses we discuss the project in general terms.
5) Partners – again under NDA – I am thinking specifically of companies like Brandenburg who would like to acquire our technology. We are also looking two major waste management businesses. This approach fits in with the disruptive technology approach – These companies are already on the ground on a regular basis with a big customer base and looking for add on services.
6) Investors – the need for NDA’s is arguably less with professional investors and institutions however I have asked all to sign NDA’a and all of them have without question. The level of dissemination involved here is actually quite challenging as the majority of them are not scientifically trained so part of the presentation has to be educational so they appreciate the level of innovation that PiedPiper project opportunity is bringing to the market.
7) Companies wishing to acquire / purchase the rights / technology. There are a number of companies in this section – for example UK and a European / North American based businesses. All members of PiedPiper have been briefed not to divulge any information without referring the company to me as coordinator. I have had a short but interesting conversation with Orkin specifically about the EU market.
8) Internationally – I have had discussions with the NZ pest control company and also the Australian pest control company have indicated a willingness to enter into a purchase agreementwhen they can get a regulatory approval
9) Biotronics have spoken to a number of Business development groups, regional and national RDA’s as well as a number of Local Enterprise Partnerships and councils. Spanish based RDA and a French Science / Technology Park. We will be arranging to deliver presentations to a number of companies and investors in the near future.

Exploitation of the PiedPiper project will continue to be carried out now the main patent application has been filed and there are a number of publications and articles on the project now in the public domain. The most accessible of which is a news article by the BBC News which was aired in the West Midlands on 2.8.2014 and a number of other regions during the following week. It is on the BBC News Environment website - the link for which is: http://www.bbc.co.uk/news/uk-england-birmingham-25575581
There is also a link to a Radio 5 Live interview with Dr Ingham at: https://connect.innovateuk.org/documents/596725/10315558/Dr+Ingham+Pied+Piper+Radio+5+live++2_01_14.wav/9ec9e3bf-af1a-4023-8a36-c35b6352507b
We have also been features in The Shooting Times and Country Magazine: http://www.shootingtimes.co.uk/news/541894/Scientists_develop_a_new_quotrat_sprayquot.html
We still have a number of magazines yet to publish
These are: New Scientist
Laboratory News
Pest Magazine
Pest Control News
NPTA – National Pest Technicians Association
And finally: Nature – the premier environmental magazine / journal.
In terms of academic papers we have the following wither published or in process:
Burgos - Dr Esther Arnaiz: Solid Phase Micro Extraction (SPME) and Has Chromatography – Mass Spectrometry (GC_MS) for the Determination of Pheromones in Mice Urine.
Dr Roberto Quesada and Dr Esther Arnaiz: Detection of small Volatile Organic Compounds in Rodent Urine using SPME, GC – MS.
The Burgos papers and posters were presented at Spanish Chemical Society Meetings.
With reference to the toxicology work Aston have the following documents in process.
1).Title: A review of cholecalciferol toxicity
Authors: Dr. A. Ingham and Dr. J. Davies
Journal: Critical Reviews in Toxicology
Status: Submitted to journal for review
2). Title: in-vivo in-vitro model for rat skin uptake of cholecalciferol
Authors: Dr. A. Ingham and Dr. J. Davies
Journal: International Journal of Pharmaceutics
Status: Submitted to journal for review
3). Title: Efficacy and toxicology evaluation of a transdermal cholecalciferol rodenticide
Authors: Dr. A. Ingham, Dr. J. Davies and Dr. M. Wei (Cellvax)
Journal: Toxicology and Applied Pharmacology
Status: Writing
4). Title: Evaluation of pheromone lures for rodent management
Authors: Dr. A. Ingham, Dr. J. Davies and Dr. R. Quesada
Journal: Applied Animal Behaviour Science
Status: Data Acquisition/writing
5). Title: Gasket compatibility for canned a transdermal rodenticide formulation
Authors: Dr. A. Ingham, Dr. J. Davies and T. Huurmann
Journal: International Journal of Pharmaceutics
Status: Writing
FP7 participants are required to use the generated results, and to this end, exploitation issues need to be planned. Each partner is responsible for using the foreground it owns, and for ensuring that such foreground can be used by other parties, keeping in mind that this use should not jeopardise the protection of the foreground, nor prevent partners from enjoying access rights to the background and foreground of the consortium. PiedPiper is no exception and we have a clearly defined agreement that detail the rights and licence arrangements. Foreground that can be applied industrially or commercially must be effectively protected. For this reason, the exploitation of the foreground, which usually implies its disclosure to the public, should not take place until appropriate steps are implemented to protect such foreground. All content of this Section XXXX therefore remains confidential until IPR protection is secured and the consortium agrees upon the disclosure of the results.
4.2.1 Description of the PiedPiper technology.
A more appropriate title might be “What has been achieved during the project”.
Pest control to limit the number of rats and mice living alongside mankind has been practised since man developed communes and centralized living areas (villages, towns and cities). Initially the control was necessary due to the rodents eating the food stores or damaging growing crops but as man developed the disease transmission risks from rodents became apparent. The PiedPiper project was highly ambitious when it was proposed and involved three essential technologies to be developed and then integrated into a useable pest control system.
1) A new method of applying the toxin formulation. There are few or no peer reviewed papers on the dermal penetration of the rat dermis with toxins let alone the use of a vitamin toxin.
2) We have developed a new method of attracting the rodent to the PCD to replace the use of food baits – most of which are wasted. The attractants were extracted from rat urine using HPLC technology. This is the first time this approach has been used in this field. We identified some 31 compounds for screening from this work.
3) The key developments in PiedPiper that will give us a marketing edge are that we have a new method of controlling and delivering the toxin and attractant in a pest control device.
PiedPiper aimed to develop a system to improve the control of rodents (rats and mice) for the benefits of society and to reduce the disease risks to humans. In agriculture, the aim is to protect food storage facilities such as grain silos. Over the recent years there have been numerous reports about food security and bio-security – PiedPiper specifically addresses these issues. This protection of food also extends to food warehouses and distribution centres from farmer through to consumer.
What PiedPiper has developed and what we can deliver for the improvement of people living in the EU:-
• Vitamin D3 is a well-documented safe molecule for humans and other species. The happy coincidence is that rats, however, are super sensitive to it.
• removal of the risk of secondary kills in birds of prey, dogs, cats and humans
• Low carbon footprint – multi-shot refillable canister
• single shot of toxin – stops resistance against the toxin as we have seen from the multi-feed warfarin products – no super rats from our toxin
• Time to death 57.4 hours (average) with no signs of poisoning up to 48 hours. The humanity index target was achieved with typically less than 12 hours of discomfort prior to death compared to 6 weeks or more with the warfarins.
The trials within the PiedPiper project have used a number of rats, with tests for example to determine the optimum dose level and ensure we had identified the correct formulation. Experiments were undertaken using facilities at University of Aston and Cellvax Pharma in Paris All trial work was done in accordance with EU and OECD regulations.
Post-mortem studies on the treated rats were undertaken to ascertain the route to death for these animals. The work produced impressive results, which conclusively indicates the changes that occur in the hours from application to death of the rats. These results clearly confirm the general stakeholder needs and the feedback from the pest control industry indicates a clear need for the PiedPiper technology as soon as possible, based on its safety and ease of use, compared to the current registered products. The 2014 ban of warfarins is seen by the industry in two ways – 1) with anxiety – will they have any other products to use on an ongoing basis? 2) If the industry sees a viable alternative it will be welcomed and used. Efforts, therefore, were made to design a system that is user friendly, safe to handle and effective.
Our original project development was a simple approach of a plastic PCD and a plug and play aerosol –a lot safer than the current method of pouring toxins out of bags by operatives with the risks of inhalation or spillage. The very low solubility of vitamin D3 also negates virtually any risk of aquatic ecosystems contamination
One of the big developments in PiedPiper was when we found that rather than needing 5 ml to deliver a toxic dose we should be able to do it with 1 ml of toxin. The solvent solubility in our chosen formulation is much superior to our pre project calculations. This gives us the opportunity to increase the number of doses in a canister. Again, the advantages are to further reduce the carbon footprint per kill and reduce the costs, as we only need 20% of the number of canisters
Table 1: Specifications of the Pest control device technology PiedPiper.
Item Technical specifications
Toxin formulation
Combination of vitamin D3 and dermal Penetrant formulation.
Pest Control Device
Dimensions200 x 250 x 400 mm
Construction: Plastic
Sensors – Infra red.
Aerosol for spraying rats.
Aerosol canister
250 / 330 lacquered mild steel with Teflon fittings to resist corrosion from the dermal Penetrant.
Attractant Extracted from rat urine for trials or synthesized In the laboratory for commercial use.

Pre-Production Prototype of the PiedPiper Pest Control Device (PCD)
This configuration of the pest control device is designed primarily for trial work as well as to test the layout and prove the component technology. It allowed us to carry out trials on
1) The PCD operation and layout.
2) The toxin formulation on the rats.
3) The attractant compounds from University of Burgos.
4) Aerosol canister efficacy in the pre-production prototype.
The next stage is to concentrate on a more aesthetically pleasing unit that will be angled towards not just optimal efficacy but also being a commercial product.
This project was very ambitious at the outset and whilst most of the techniques and processes were already in laboratory and design practice the applications were new.
Our dermal penetration approach was new as we were looking at a very new approach – One shot -= One Kill! Our approach was to change from multi-feed baits, to delivery of a new toxin into the bloodstream of the rodent – not just to poison it as current products do but to guarantee a kill every time.
Keep it simple - The infrared sensors in the unit had far too high a power requirement it was redesigned, in the new system the units are switched on for 5 milliseconds and then switched off for 950 milliseconds, this extends battery life from two weeks to over a year.
The Aerosol technology we are using is from the global aerosol valve manufacturer. Dr Quesada and his colleagues in Burgos took on a huge task to identify the attractant compounds from rat urine. This approach is new to this sector and the 38 compounds isolated and identified were screened then trialled by Aston / Burgos and the post-mortem and histology work done by Cellvax.
4.2.2 Protection and Exploitation of Intellectual Property Rights.
Different types of intellectual property were covered in our discussions including patents, registered designs, copyrights, design rights, trademarks, know-how-secret or proprietary information. In addition details on patents, patent co-operation treaties (especially for SMEs to delay costs), benefits of patent searches, tax benefits considerations (e.g. UK Patent Box tax relief, Ireland 10% flat rate of tax on IP , details on copyright, trademark, registered and unregistered designs were also considered.
Knowhow is considered as the most important form of protection and transfer of technolog, which can include drawings, designs, instructions, tools, software, formulae, or materials. Patent attorneys were asked to offer strategic advice on potential changes to pending patent applications, timing of initial application, timelines and schedules, costs, levels of detail etc.
We covered a number of the exploitation agreement issues in our discussions – we considered two case studies, ”A Patent Exploitation Criteria” and ”A Study on the Patent Exploitation and Management Best Practice Model for Japanese Small and Medium Enterprises”.
The result of these studies makes it clear that input from each of the SMEs is needed in order to make meaningful decisions in relation to the Exploitation Agreement. In terms of the commercialization of the results of this project, it has already been agreed at our Kick off meeting and M9 meeting that the SMEs waited until the results of the project became clearer, before developing commercially exploitable IP as well as deciding on intellectual property protection issues. Having done this we have now had patent attorneys draw up the Toxin Formulation patent and that application has been submitted.
Market Potential and Business Plan.
Description of the market
The global pest control market runs into multi Billions of Euros annually. In the EU according to the EPCA (European Pest Control Association) it is currently recorded at 3.5 billion euro’s for its members and they estimate 3.75 billion euro’s for the entire EU. The Japanese market has less than a third the population of the EU but the pest control market is slightly larger.
The US and Pacific markets are more fragmented and less well documented but figures from a New Zealand company estimate the market in New Zealand is 65,000,000 Euros alone in contracts from DOC (Department of Conservation) and a further 35,000,000 in domestic / commercial customer services.
As is well documented the EU is to phase out warfarin toxins in pest control in 2014 which will leave a limited range of snap traps and electronic methods of pest control. Second Generation Anti-Coagulants (SGAC’s) are still registered in the EU until at least their review in 2015/16. The pest control organizations and companies are lobbying the EU and governments to be allowed to use their SGAC’s more widely and at higher doses. This is an environmental disaster waiting to happen and the use of PiedPiper technology will prevent this from happening. Our (vitamin D3) toxin has now been proved in trials to be a viable toxin for the marketplace with the following benefits :-
1) Low toxicity to humans, this gives safety and security of handling.
2) Single shot delivery not multi-feed where tolerances can build up as with the warfarins. One shot = one kill.
3) Safe to other wildlife – cats, dogs and birds of prey who may consume poisoned carcasses.
4) Effective – rapid time to death (currently 48 hours max) as compared to up to six weeks with warfarins.
5) Low carbon footprint – PiedPiper PCD will need less servicing than bait stations (rats currently need to eat around their weight in toxin block /grain mixes to kill them). Our system requires 1ml = 1gm. We also offer less time travelling to rodent sites as they do not need the same level of visits.
The PiedPiper finished product is being specifically designed and developed to meet the needs of the pest control industry so that it can keep the people of the EU safe from rats and the disease risks they pose.. It will allow them to use our product with ease and efficiency and most of all safety. Most pest control companies in private conversations are not that environmentally friendly so a product that is environmentally friendly – stopping secondary kills for example will be well received. We have had a number of communications with the environmental agencies including the DG for the Environment. Finally, a SWOT analysis was carried out (Table 4 below) based on key findings and conclusions of the market research, enabling the consortium to identify the main Strengths, Weaknesses, Opportunities and Threats of the proposed research. This analysis will be the guideline for the remainder of the project both in terms of the research and development work and direction as well as for the design and implementation of dissemination and exploitation activities to promote the future uptake of PiedPiper’s PCD
1) Low toxicity to humans, this gives safety and security of handling.
2) Single shot delivery not multi-feed where tolerances can build up as with the warfarins. One shot = one kill.
3) Safe to other wildlife – cats, dogs and birds of prey who may consume poisoned carcasses.
4) Effective – rapid time to death (currently 48 hours max) as compared to up to six weeks with warfarins.
5) Low carbon footprint – PiedPiper PCD will need less servicing than bait stations (rats currently need to eat around their weight in toxin block /grain mixes to kill them). Our system requires 1ml = 1gm. We also offer less time travelling to rodent sites as they do not need the same level of visits.
The PiedPiper finished product is being specifically designed and developed to meet the needs of the pest control industry so that it can keep the people of the EU safe from rats and the disease risks they pose.. It will allow them to use our product with ease and efficiency and most of all safety. Most pest control companies in private conversations are not that environmentally friendly so a product that is environmentally friendly – stopping secondary kills for example will be well received. We have had a number of communications with the environmental agencies including the DG for the Environment. Finally, a SWOT analysis was carried out (Table 4 below) based on key findings and conclusions of the market research, enabling the consortium to identify the main Strengths, Weaknesses, Opportunities and Threats of the proposed research. This analysis will be the guideline for the remainder of the project both in terms of the research and development work and direction as well as for the design and implementation of dissemination and exploitation activities to promote the future uptake of PiedPiper’s PCD
1) Low toxicity to humans, this gives safety and security of handling.
2) Single shot delivery not multi-feed where tolerances can build up as with the warfarins. One shot = one kill.
3) Safe to other wildlife – cats, dogs and birds of prey who may consume poisoned carcasses.
4) Effective – rapid time to death (currently 48 hours max) as compared to up to six weeks with warfarins.
5) Low carbon footprint – PiedPiper PCD will need less servicing than bait stations (rats currently need to eat around their weight in toxin block /grain mixes to kill them). Our system requires 1ml = 1gm. We also offer less time travelling to rodent sites as they do not need the same level of visits.
The PiedPiper finished product is being specifically designed and developed to meet the needs of the pest control industry so that it can keep the people of the EU safe from rats and the disease risks they pose.. It will allow them to use our product with ease and efficiency and most of all safety. Most pest control companies in private conversations are not that environmentally friendly so a product that is environmentally friendly – stopping secondary kills for example will be well received. We have had a number of communications with the environmental agencies including the DG for the Environment. Finally, a SWOT analysis was carried out (Table 4 below) based on key findings and conclusions of the market research, enabling the consortium to identify the main Strengths, Weaknesses, Opportunities and Threats of the proposed research. This analysis will be the guideline for the remainder of the project both in terms of the research and development work and direction as well as for the design and implementation of dissemination and exploitation activities to promote the future uptake of PiedPiper’s PCD
1) Low toxicity to humans, this gives safety and security of handling.
2) Single shot delivery not multi-feed where tolerances can build up as with the warfarins. One shot = one kill.
3) Safe to other wildlife – cats, dogs and birds of prey who may consume poisoned carcasses.
4) Effective – rapid time to death (currently 48 hours max) as compared to up to six weeks with warfarins.
5) Low carbon footprint – PiedPiper PCD will need less servicing than bait stations (rats currently need to eat around their weight in toxin block /grain mixes to kill them). Our system requires 1ml = 1gm. We also offer less time travelling to rodent sites as they do not need the same level of visits.
The PiedPiper finished product is being specifically designed and developed to meet the needs of the pest control industry so that it can keep the people of the EU safe from rats and the disease risks they pose.. It will allow them to use our product with ease and efficiency and most of all safety. Most pest control companies in private conversations are not that environmentally friendly so a product that is environmentally friendly – stopping secondary kills for example will be well received. We have had a number of communications with the environmental agencies including the DG for the Environment. Finally, a SWOT analysis was carried out (Table 4 below) based on key findings and conclusions of the market research, enabling the consortium to identify the main Strengths, Weaknesses, Opportunities and Threats of the proposed research. This analysis will be the guideline for the remainder of the project both in terms of the research and development work and direction as well as for the design and implementation of dissemination and exploitation activities to promote the future uptake of PiedPiper’s PCD
SWOT analysis of the Pied Piper project
Strengths
Fully integrated plug and play unit, the only additional requirements being occasional changing of batteries – which may be rechargeable.
Low carbon footprint – saves on visits.
Multi-shots - highest kill index of any toxin – every shot on a rat will kill
Safe to other wildlife – birds of prey, cats, dogs etc.
Safe to humans
Pest Control device made of recycled recyclable plastics – low carbon footprint.

Weaknesses. At this time, we do not have a finished commercial product but have all the technology developed to produce a final product.
Regulatory approvals will be required.
Big business – we are a very small player in a very large market so we will tread on corporate toes. It is clear from discussions during this project that there are a few companies that would like to “own” the PiedPiper rights, Some that would like to partner us and a few who are totally opposed to what we are going to do the market.

Opportunities
Readiness for investments to make improvements in the industry. Following the announcement of the warfarin ban, the European recession, the requirement to cut costs and be more efficient the industry needs a new product.- and PiedPiper is a perfect fit.
Global multi-billion Euro market in pest control

In markets where warfarins are not to be banned, our delivery system could be licenced to give safer usage of existing product.
Opportunity to raise the profile of pest control market – initially we thought to concentrate on the professional market but once established we could move into the domestic market selling online and from DIY chains Generally we have seen a keen interest in finding out more about the project and requests for samples or product when it is available
Current lack of alternatives to the warfarin products for general use is not well received in the marketplace - offering a greener, low carbon footprint, safe product will be met by consumers with enthusiasm.
We regard to this project we have generated a lot of data and information providing a great dissemination opportunity to raise awareness among the pest control industry of the significant advantages of the PiedPiper technology.
We have had approaches from a number of Specialist Pest Control companies for partnership or development deals – these include – BASF (Germany – Head of R and D), Brandenberg, Pelgar, Orkin (USA), Rentokil UK, KillGerm Europe and an offer to talk to the Welsh Assembly about manufacturing in Wales.
Post the BBC News item we have had a lot of additional coverage and it is clear that the vast majority of people are 100% behind our technological approach.
Threats The Industry in general was unfamiliar with PiedPiper technology and the use of Vitamin D3 as a toxin. As a disruptive technology our plan is not necessarily to market via the existing pest companies but look at options like Viola (Waste Management) and Biffa Waste Management – these companies are working in rat-infested environments 24 / 7 / 365.
Negative marketing from competitors – we have heard the odd comment about the technology does not work and they are too underfunded to get to market as the regulatory costs are too high.

Regulatory barriers to markets.
4.2.3. Unique selling points
The laboratory results confirm that we have developed a superior performance pest control device that is a very new concept to the industry. We aim to emphasise the following USP’s for PiedPiper:
1) Low toxicity to humans giving safety and security of handling.
2) Single shot delivery not multi-feed where tolerances can build up like the warfarins.
3) Safe to other wildlife – cats, dogs and birds of prey who may consume poisoned carcasses.
4) Effective – rapid time to death (currently 57.4 hours) as compared to up to six weeks with warfarins.
5) Low carbon footprint – needs less servicing than bait stations (rats need to eat around their weight in toxin block /grain mixes to kill them). Less time and travelling to sites = reduced carbon footprint.
Environmental energy impact savings in conjunction with an increased safety and efficiency of use would also be additional strong selling points for PiedPiper. In terms of pest control usage it will significantly cut the time to death compared to currently licenced products and thus reduce the suffering of the animals. Our laboratory results already indicate that significant dose rate improvements may still be possible but further refinements in the formulation may not reduce the time to death as we believe that this is optimized at 48 hours without suffering then a further 10 hours to death.
4.2.4 Outline of an initial business plan
How can the intellectual property best be protected in the interests of the SME consortium? It is important to plan how the technology can be brought to market, based on the fully functional prototype the consortium discussed and reviewed the possibilities of different business models during the M24 meeting. Several options for bringing the technology to the market are possible. This is not a product where each partner could market the technology individually as the SME’s skill sets are very diverse – as required by the FP7 project rules and the project needed the diverse skills to develop the technology.
Our Consortium Agreement laid out that licence fees will be paid to two SME’s and Summit Europe will supply their valves to the manufacturer of the aerosols. This has been changed due to the liquidation of Helios in March 2013.
In terms of business models, we have a number of options:
1) Biotronics take the product to market on behalf of the SME’s
2) A custom European pest control company is set up
3) The technology is sold / licensed to either a partner pest control company or a full technology sale made.
PiedPiper Business Plan Options.
Having reached the point of completing a successful research project to maintain the momentum of development we need to have a plan for dissemination, development and exploitation of the PiedPiper technology.
All the current individual members of PiedPiper will be involved in the second FP7 grant - PiedPiper DA in one way or another.
What we need to disseminate to the industry and the public across Europe is that PiedPiper is a humane, revolutionary technology for the Pest Control sector that safely overcomes the issues of tolerance of the multi-feed toxins. Furthermore it has the power to be a disruptive innovation technology that gives us the opportunity to reshape the pest control industry. The advantage of the technology is that we are not locked in to the traditional supply and distribution chains and thus can pick the most advantageous and cost effective routes to market to benefit all of the members of the EU – after all it is public funds that promoted this project.
There are a number of routes to market that we can use to develop the PiedPiper technology for the European market. These are as follows:
1) Licence
2) Partnership Deal
3) Sale to larger company
4) Investment company route to market
5) Investment company / PLC
6) Sale to larger company with licence or royalties
7) Franchise
8) Foundation.
It is difficult to finalize these options at this point. One thing that is certain is that the PiedPiper team are committed to promoting the advantages of the technology here in the EU and globally. We look forward to further promotion and demonstrations of the product and developing further environmentally features. One of the key features of this technology is what we are going to be able to do with regard to making the EU safer from Rodents – thus reducing health risks, contamination of food supplies, enabling easier maintenance of restraints, Hospitals and other buildings. It will take a little time to rollout the technology but ultimately we will be able to bring under control the rodents currently resistant to the current toxins and reduce their numbers. Once reduced the ongoing control will be a lot easier and we will not get a repeat of the toxin resistance scenarios. The reduced numbers will also bring other benefits for example reduced pest control costs for councils, and other bodies involved in pest control work.

List of Websites:
piedpiperglobal.com
biotronics@cix.co.uk

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BIOTRONICS LTD
United Kingdom
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