Wednesday, October 28, 2015

Trace Environmental Substances Showing up as Post-Race “Positives” : What every horseman needs to know about being wrongly accused.


by Clara Fenger, DVM, PhD, DACVIM; Steven Barker, PhD; Keith Soring, DVM; Lee Shalgos, Esq.; Thomas Tobin, MRCVS, PhD, DABT.

The unveiling of the National Uniform Medication Program (NUMP) has come about with much fanfare and publicity; many states, such as the Mid-Atlantic States, signed on quickly while others have been more reticent to come on board.  The scientific basis for the thresholds which are supposed to permit appropriate use of the somewhat restricted list [27] of therapeutic medications while preventing unnecessary and excessive overmedication have hardly been forthcoming, and many hold-out states recognize these problems.  Despite the hurdles which NUMP faces from states unwilling to adopt such restrictive plans and from the internal deficiencies with the “Uniform Program”, such as insufficient scientific evidence, the over-regulation of therapeutic medications is not the only drug related obstacle which racing must face.

Substances used by humans, such as caffeine, cocaine and methamphetamine, commonly prescribed medications  for humans, such as tramadol and anti-anxiety drugs, and even over the counter medications like Aleve and ibuprofen have all come up as trace level “positives” in post-race samples in horse racing.  The biggest question surrounding these “positives” is:  Are these inadvertent environmental exposure, which horses picked up from eating contaminated hay, perhaps the result of a groom relieving himself innocently in a stall, or are they evidence of nefarious activity, forensic evidence of an attempt to steal a horse race?

In the early 1990’s, as the use of crack cocaine rose to a crescendo in the human population, there was a simultaneous rise in the general exposure of the population to trace levels of cocaine.  In fact, there are estimates that 90% of the paper currency in circulation is contaminated with measurable levels of the illegal substance.  People have even been wrongly accused of drug charges as a result of drug sniffing dog detection when they were only guilty of carrying large amounts of paper currency.  Our paper currency is so contaminated with drug residues that a confirmatory level of the major urinary cocaine metabolite, benzoylecgonine (BZE) at 100 ng/mL in urine is considered by the federal government to be a “negative” in that it is a forensically insignificant test.  Not everyone who handles money will test approaching this level:  this is one of the key points of environmental contamination related “positives”:  they strike randomly, and a number of important factors must align, including exposure to a source, individual metabolism and timing of the sample collection.   

The need for regulatory “cut-offs” for a number of environmental substances has been recognized by a number of states, which have put such cut-offs in place.  For example, in Ohio, there is a level for BZE (150 ng/mL in urine) and morphine (50 ng/mL in urine), the BZE “cut-off” being similar to the earlier  thresholds adopted by the federal government for BZE  as environmental contamination, as detailed in the 2012 National HBPA book on World Rules for Equine Drug Testing and Therapeutic Medication Regulation.

The potential for environmental residues of therapeutic substances to give rise to equine “positives” has been demonstrated repeatedly in the scientific literature.  When naproxen (Aleve ®) was administered to horses for 10 days, they continued to show measureable urine levels for over 45 days.  These researchers interpreted this prolonged elimination time  as being  due to stall contamination  with the medication and, consistent with this interpretation, when a horse which received no naproxen was placed in the stall of a naproxen treated horse, this horse  subsequently showed “positive” for urinary levels of naproxen for three days.  This occurred despite the fact that the stall and manger of the stall had been “thoroughly” cleaned.  Similar findings have been reported with flunixin (Banamine ®).  When untreated horses were placed in stalls of horses which had been treated with flunixin for 14 days, the untreated horses tested positive for 2 of the following 14 days.  Similar to what has been identified with BZE in humans, the appearance of an environmental contamination positive can clearly be associated with exposure to environmental residues.  In another study, isoxsuprine could be identified from the floor, walls manger, and even the cobwebs in the rafters of a stall of a horse which had received a therapeutic regimen of the substance, which was then discontinued for three weeks pre-race. A number of other studies have found similar results for a variety of drugs.

Key Factors Influencing a “Positive” from an environmental exposure

What are the key reasons that some substances can remain in the environment and therefore pose a risk for inadvertent environmental exposure to the competitive equine athlete?  There are three key criteria which must be met in order for a substance to represent a high risk.  The first criterion is that the substance, like isoxsuprine, must be chemically stable in the environment.  Once it is introduced into a stall it remains there, and hangs around waiting throughout the stall, including apparently in the cobwebs.  For an environmental contaminant be a significant problem, it first needs to be chemically stable and therefore persist in the environment.



The second point is that the contaminant must be easily absorbed, which usually means orally absorbed.  For an environmental substance to give rise to drug testing problems, it must be able to get into the horse from the environment.  Oral absorption is likely route number one, and we know that isoxsuprine is well absorbed orally.  This is more problematic than the aforementioned incidental exposure of humans to cocaine tainted currency:  horses, especially intact males, are well known to seek out urine in stalls, smelling and tasting the waste from any previous occupants to identify who may have been in that stall before them.  This is an animal behavior unparalleled by humans.



Next comes a very interesting point; although isoxsuprine is well absorbed orally, effective blood levels of isoxsuprine after oral administration are difficult to achieve.  Orally absorbed Isoxsuprine is so rapidly and effectively metabolized/glucuronidated on its first pass through the liver that it fails to rise to effective plasma concentrations after oral administration.  Orally absorbed isoxsuprine is essentially immediately glucuronidated, or changed into an inactive metabolite, while passing through the liver, and it is then shipped as the metabolite to the kidney for excretion.  This feature of isoxsuprine yields the highest known concentrations of any equine drug metabolite, peaking at 700,000 ng/mL.; given that about a 700 mg IV dose of isoxsuprine produced this urinary level of isoxsuprine in urine, a 1 mg dose could produce a 700ng/ml urinary concentration, readily explaining the ability of minute amounts of environmental isoxsuprine to produce low nanogram/ml urinary isoxsuprine glucuronide “positives” . 



To summarize, isoxsuprine is chemically stable in the environment, it is well absorbed orally and is, for all practical purposes, immediately converted into its glucuronide metabolite and excreted at very high concentrations in equine urine.  Take home message:  very small environmental traces of isoxsuprine passed in the urine of one horse, and lingering in its stall will result in sufficient isoxsuprine glucuronide in the urine to trigger a “positive” call for isoxsuprine in any untreated horse which subsequently inhabits the stall.

So the rules for a problem environmental substance are:  (1) it must be chemically stable in the environment; (2) it should be well absorbed orally; (3) it should be excreted in relatively high concentrations in the urine.  Any substance with these chemical/biological characteristics has the potential to be an environmental substance “positive” problem in equine drug testing, as we will see.

Tramadol

Substances  that become widely used in humans for foodstuff [caffeine] recreational [cocaine methamphetamine] or medical use [Ritalin], or as prescription pain medications, such as Tramadol, may well spill over into the equine world as trace environmental substances and  show up, usually at trace levels,  in racehorses.   In contrast to its therapeutic effectiveness as a painkiller in both humans and small animals, Tramadol in the horse has failed to show any significant analgesic effects in horses at similar doses.  At very high doses, exceeding 10 times the effective dose in humans, by weight, Tramadol can induce analgesic effects that may last for less than three hours.  This lack of efficacy in equines appears to stem from the rapid inactivation of the active primary metabolite, 4OH-tramadol, by glucuronidation, a unique feature in this species.  Nonetheless, this inactivated metabolite is detectable in the horse’s system for a long time after the last administration.

Tramadol is clearly stable in the environment; one environmental study looked into the fate and potential impacts of Tramadol after its introduction into water.  It was found that neither tramadol, nor its derivatives formed from exposure to light were significantly biodegradable according to standard test guidelines.  They simply remain in the water forever.  In Europe, Tramadol was #3 on the list of pharmaceuticals found in wastewater, ranked by median concentration, with the highest wastewater concentration reported being 1166 ng/L or 1.166 ng/ml.  Similar to flunixin and isoxsuprine, tramadol is highly persistent in the environment, and could readily serve as a substance of incidental environmental exposure in the horse.

On July 11th 2013 at Hoosier Park Indiana the Harness horse “Justice Jet” yielded a positive for “Tramadol”.  We assume that the actual chemical nature of the analyte was O-desmethyltramadol and the concentration identified was not available to us at the time of writing.  At the time of the identification Tramadol was classified by ARCI as a Class 2, Penalty Class A substance.  However, the trainer involved was apparently highly regarded, with a reportedly 30 year unblemished medication violation record.  Additionally, the trainer was unaware of any risk factors such as individuals prescribed Tramadol in the environment of or close to the horse in question.  Given the circumstances of this case and the apparently complete innocence of the trainer, a review of the ARCI penalty classification for Tramadol was requested by the Indiana Racing Authorities.  While Tramadol is still listed as an ARCI Class 2, penalty class A substance, the Indiana authorities chose to penalize the trainer involved with a relatively “modest” 15 day penalty and a $500.00 fine, a much less severe penalty than the in place ARCI penalty guidelines would have suggested.  It would also be fair to say that in absence of an identified prescription for Tramadol in the racing environment of the horse, this case becomes a simple generic environmental exposure case, with no specifically identified source for the detected Tramadol.  

In a December 4th 2013 case before the New Zealand Judicial Control Authority for Racing, human use of Tramadol was considered to account for “exposure” of a horse to Tramadol, i.e., a reported identification in a Standardbred racehorse.  The trainer/ person responsible for the horse had been prescribed Tramadol by her doctor for back pain, and the Tramadol metabolite O-desmethyltramadol was found in a post-race sample taken from the horse.  Under the New Zealand Rules of Harness Racing, the presence of Tramadol or its metabolites is prohibited. The person responsible admitted to taking 50 mg Tramadol capsules on race nights, and the most likely cause of exposure to the horse was considered to be through contamination of her hands after taking the medication and bridling the horse.  The concentration of tramadol identified in the sample was extremely low, 100 pg/mL (0.1 ng/ml), much lower than the LOD (limit of detection) in the recently published Knych et al. (2013) report.   It is also noted that this very low Tramadol concentration was not identified in the primary New Zealand testing laboratory, but was only identified upon additional and apparently Tramadol-unrelated high sensitivity retesting of the sample by the Hong Kong Jockey Club laboratory.  The trainer in question was fined $3,300 and to our knowledge was not suspended.

In this matter the New Zealand judicial authority appears to have accepted the likelihood of inadvertent exposure of the horse to very small and pharmacologically insignificant amounts of Tramadol associated with Tramadol being prescribed for medical purposes to the trainer in question. 

Similarly, in one California Tramadol positive in a racehorse, the trainer was prescribed Tramadol for chronic back pain associated with a training accident.  This trainer’s horse had tested “clean” only ten days earlier, which underscores the random nature of the environmental positive.

Cathinone

Cathinone, a component of the synthetic amphetamine “bath salts,” has been identified in trace amounts in racehorses in Iowa.  Bath salts hit the streets in the US in 2009 as a drug of human abuse, reaching epidemic proportions by 2011 and started to show up in racehorses in Iowa in 2011.  Further complicating the interpretation of the test, cathinone can be found in urine in some testing systems after the administration of the common antihistamine, Pyrilamine (TriHist®, Anihist®), ephedrine (Sudafed®), or propanolamine.  The AniAaaaAAThe first racehorse positive was in 2011 at a level of 3 ng/mL  in urine, too low to reflect any pharmacologic effect on the animal, and consistent with other drugs of human abuse:  environmental exposure at low levels.  The trainer was summarily suspended and even handcuffed and removed from the grounds in a squad car.  Shortly thereafter, other positive tests for cathinone started to trickle in, and over the course of 70 days, there were 16 trainers involved, encompassing 51% of the barns and 5 different veterinary practices.  The sheer number of “positives”, and the low level of the drug in the animals suggested that  it was likely that this was an environmental exposure (Kind et al., 2012).

A survey was conducted to identify any commonality among the positive identifications of cathinone, and none could be identified.  Hay and several species of broad leaf plants from local farms were analyzed without success and the source of the contamination could not be identified.   Human drug testing was inconclusive as well.   However, based on the levels found in the positive tests, an environmental contamination “cut-off” was determined to be 10 ng/mL, which was adopted by the Iowa Racing Commission as a new regulatory threshold. 

The cathinone positives have slowed down in Iowa after this initial spike, but regulators remain convinced that these positives resulted from some exposure in the environment, possibly of plant origin, although simple contamination from human recreational use cannot be ruled out. 

Methamphetamine



            Another emerging substance of inadvertent environmental exposure in horses is methamphetamine.  Like many of the other substances of concern, methamphetamine has become a widespread drug of human abuse.  A recent case involving a cluster of trace concentration methamphetamine positives in Canada provides a classic example.  These events started when a Michigan trainer purchased a large used horse trailer shortly before shipping her Quarter horses to Toronto to race at Ajax downs. The three horses that shipped in this trailer raced within two days of shipping, and all three tested positive for picogram urinary concentrations of methamphetamine, while another horse, which shipped in a different trailer, also raced but tested negative.  This circumstance presents a classic "cluster" of very low, picogram concentration methamphetamine positives, two occurring on day one and one the second day post shipping, with a horse not shipped in the suspect trailer testing negative.



The Ontario investigators interviewed the trainer and as part of their review tested a number of samples from the trainer's equipment, including a sample from the manger area of the trailer.  This trailer sample turned up “positive” for methamphetamine at 22 ng/milligram, providing a clear-cut environmental source for the methamphetamine identified in the horses shipped in this trailer. The most likely interpretation of these events is that this trailer, at some time in its previous life, had housed an illicit methamphetamine laboratory, and that traces of methamphetamine remained in the newly purchased trailer and transferred to the horses during shipping to Toronto. Similar environmental related methamphetamine contamination of humans working in or around methamphetamine synthesis facilities are well understood.  What is interesting, however, is that this is the first clear-cut link between an environmental source of methamphetamine and a classic "cluster" of low concentration environmentally driven equine methamphetamine identifications.



As with isoxsuprine, we can also estimate the dose of methamphetamine required to give rise to these reported identifications.  Work by Ohio State University researchers performed in the early 1970s shows that administration of a 150 mg dose of methamphetamine (a comparable dose to the effective dose in people) to a horse will yield a peak urinary methamphetamine concentration in the region of 7,000 ng per ml.  Simple arithmetic shows that 1 µg of methamphetamine, or 1/150,000th of the effective dose investigated by the researchers in Ohio, is all that is required to yield urinary concentration of 47 pg per ML, a concentration very close to the lowest urinary methamphetamine concentration reported in this Toronto cluster, which was 56 pg per ML, or parts per trillion in urine.  Methamphetamine adheres to thhe three key factors of an inadvertent environmental exposure:  (1) it is a chemically stable substance that is (2) orally absorbed from the environment of a horse and (3) can appear in urine at extremely low concentrations, but such findings are indicative of nothing more than totally minuscule exposure of the animal to the substance in question.  Further, the pervasiveness with which methamphetamine is used as a substance of human abuse greatly increases the likelihood that it will overlap with an unsuspecting population of horses.

Other Drugs of Human and Animal Use

There has also been a recent flurry of low-level “positives” for O-desmethylvenlafaxine, the major metabolite of venlafaxine, better known as the anti-depressant Effexor, in Canada, the USA and recently in India.  As with Tramadol, routes of human contamination of the horses are thought to be the same. In a small study conducted by Canadian authorities, a horse was dosed with venlafaxine and an aliquot of the horse urine used apparently “poured” on hay and the hay placed in a “clean” stable and housing a “clean” horse, rapidly leading to the “clean” horse becoming positive for O-desmethylvenlafaxine.  Indeed, low levels of this metabolite have also been found in supplements. This is similar to the sildenafil or Viagra positives seen several years ago, where a supplement source for the drug was identified in products produced by a compounding pharmacy.

Across the US and Canada there has also been a number of very low level levamisole positives. Levamisole, an “old time” wormer approved by the FDA for use in cattle and commonly used off label for horses was originally categorized as an Association of Racing Commissioners International, ARCI  Class 4 substance, but has been reclassified as a 2 with an A penalty.  It has experienced a resurgence in usage in horses in recent as a result of its immune stimulating properties, and has been widely recommended by researchers in the field of Equine Protozoal Myeloencephalitis.  Additionally, levamisole is very environmentally stable, is routinely used in food animals and can be found in fertilizers made from their excrement.  Experiments are currently underway to determine how much of this compound is taken up into food products such as corn and whether or not this is a potential source for some levamisole positives.  Nonetheless, levamisole can be found in runoff from organic fertilizer treated fields.

The list of such instances could continue and many more cases for low level positives in racing could be cited. This is, in many cases, the result of our ever increasing ability to detect pharmacologically insignificant amounts of drugs commonly used by humans and other animals in equine samples. More and more of the positives being called approach the low picogram (1,000th of a nanogram)/ml level and are being treated by many Commissions as if they were pharmacologically effective levels normally seen immediately following administration. There is, in such cases, no distinction being made between “trace” and perhaps even “ultra-trace” levels arising from environmental sources and what a true attempt to influence performance actually requires. “Zero Tolerance” or no tolerance for the finding of a substance, regardless of the level, has once again reared its ugly head but is now armed with more sensitive equipment than ever before.

As more therapeutic medications are prescribed to humans and used in food animals, as more enter the environment of the horse by a variety of mechanisms, as more medications and recreational substances  go into our wastewater, our foods, feeds, supplements and even the air, we will eventually reach a point where the sensitivity of our testing and the pervasiveness of these drugs will cause ever greater numbers of positives for the racing industry.  If we fail to address these facts the consequences are an ever increasing suspicion of corruption in racing, from the calling of drug “positives” at irrelevant levels which inevitably erode confidence in the integrity of the industry.  The attempts by several of the controlling associations to subsequently regulate common therapeutics by placing thresholds on useful veterinary products at the same level as may occur from contamination will only add to this problem. It is possible that their attempts will only result in the opposite of what they desired.

Conclusions:

For reasons that are unclear the RMTC is uninterested in setting thresholds/cut-offs for inadvertent environmental exposure, despite the obvious need and at times specific requests for such guidance by regulators and horsemen’s organizations.   It is incumbent upon the regulators that police racing to distinguish between unavoidable trace level environmental exposure with no effect on the animal or the competitive event and no intentional malfeasance.  Innocent and unavoidable environmental sources should be considered in every instance in which low levels of substances are identified in post-race samples.  Identification of trace levels of substances of human abuse should be carefully investigated, and policies should be enacted to prevent the improper penalization of innocent horsemen.  In fact, in New York, the Court of Appeals has ruled that testing laboratories for humans could be held liable for calling positives on drug levels consistent with environmental contamination.

Guidance for Horsemen:

The primary source of environmental contamination positives in racehorses is exposure to humans who have handled or are taking prescription medications or illegal substances.  The best defense is careful oversight of your shed row.  If any grooms are suspected of illegal drug use, drug testing, with hair testing is by far the best option followed, where necessary, by removal from the care of the horses is the best option.  Any grooms taking prescription medications should be warned to wash their hands thoroughly between taking their medications and handling horses.   Tongue ties are of particular importance.  Use only new tongue ties on race-day, and do not allow grooms to carry them in their pockets, where they may be commingled with prescription pill fragments.   Carefully admonish grooms and riders NOT to urinate in the horse’s stalls.  Even over the counter pain medication, like Aleve® have been implicated in positive tests as a result of careless urination by a groom in a horse’s stall.

Horsemen and veterinarians should be cognizant of the source of their medications and supplements.  Positives may result from the use of supplements and compounded materials prepared and sold by less reputable compounding pharmacies, containing unidentified drugs.  Often, claims that sound too good to be true are exactly that. 

As the medication rules become more onerous, horsemen must be ever more proactive in controlling the environment of their charges.  Even when we have controlled everything in our shed rows, there will still be inadvertent environmental contamination positives, because we cannot control the ship-in barn.  Horses live in barns and not maximum security facilities.  We can only hope that the regulatory pendulum swings back towards reason, and our regulators can determine a way to avoid unfair penalizing of innocent horsemen, much like the justice system ultimately did the right thing by the unfairly convicted parolees of the 1990’s.

Suggested additional reading:



Kind AJ, Soring K, JD, Brewer K , Eisenberg, R., Hughes CG, Hartmann-Fishbach, P and Tobin T (2012) Cathinone and Related "Bath Salt" Substances - Detection in Equine Urine and Potential Sources, In press, The 19th International Conference of Racing Analysts and Veterinarians, University of Pennsylvania, Philadelphia, Pennsylvania, September 2012.


Wennerlund I, Ingvest-Larson C, Kallings P, Fredrickson E, Bondesson U.  2000.  Pharmacokinectics and urinary excretion of naproxen after repeated oral administration in the horse.  In RB Williams, E Houghton, JF Wade (Eds) Proceedings of the 13th Annual Conference of Racing Analysts and Veterinarians (pp. 195-200).  Cambridge, UK.

Norgren A, Ingvest-Larson C, Kallings P, Fredrickson E, Bondesson U.  2000.  Contamination and urinary excretion of flunixin after repeated administration in the horse.  .  In RB Williams, E Houghton, JF Wade (Eds) Proceedings of the 13th Annual Conference of Racing Analysts and Veterinarians (pp. 377-380).  Cambridge, UK.

Russell CS, Maynard S.  2000.  Environmental contamination with isoxsuprine.  In RB Williams, E Houghton, JF Wade (Eds) Proceedings of the 13th Annual Conference of Racing Analysts and Veterinarians (pp. 377-380).  Cambridge, UK.

Knych HK, Corado CR, McKemie DS, et al. 2013. Pharmacokinetics and pharmacodynamics of tramadol in horses following oral administration. J Vet Pharmacol Ther 2013;36:389-398.

Loos R, Carvalho R, Antonio DC, et al. 2013. EU-wide monitoring survey on emerging polar organic contaminants in wastewater treatment plant effluents. Water Res 47:6475-6487.

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