What Makes A Positive Test: An Explanation Behind The Figures And Thresholds by Dr. Stacey Oke|01.05.202101.12.2021|1:02pm3:05pm Positive post-race tests have long been a source of fascination and discussion for horsemen, veterinarians, and fans of racing. But for those of us without an advanced chemistry degree, it can sometimes be unclear what those tests are meant to look for, and what makes a test “positive.” Veterinarians administer medications to Thoroughbreds in accordance with withdrawal guidelines provided by rulemakers. The Association of Racing Commissioners International (ARCI) provides a set of recommended guidelines for what should be considered a “positive” test, and when doses of certain drugs can safely be given to comply with those guidelines. If these guidelines are followed, experts say the owner or trainer who approved the medication should not be overly concerned about having a “positive” test after a race. Let's take phenylbutazone (widely referred to as “bute” for short) as an example. This is one of the most commonly used medications in Thoroughbred racehorses due to its anti-inflammatory properties. The suggested ARCI dose of bute for a 1,000-pound horse is 4 milligrams of the drug per kilogram of the horse's weight (mg/kg) when given intravenously (IV). For the average horse that works out to a 1.8-gram dose or 1,800 mg. According to the guidelines, this dose should not be administered less than 48 hours before a race. “The ARCI threshold or 'cut off' value in blood is 0.3 micrograms/milliliter of blood. This is a small amount: 30 one-millionths of a gram of bute in each milliliter,” explained Mary Scollay, DVM, the Executive Director of the Racing Medication and Testing Consortium (RMTC). (A milliliter is one one thousandth of a liter.) A horse has approximately 50,000 milliliters of blood, so he can have up to 15,000 micrograms (mg) of bute total in his blood at the time of testing. This is equivalent to 15 mg or 0.015 g. If 1.8 grams of bute were administered 48 hours before testing, what is the amount of bute actually remaining in the horse 48 hours post-administration? That answer depends on how slowly or quickly each horse metabolizes the bute. As a first approximation, let's assume the half-life of bute in the horse is about 7.22 hours, based on available research. This means that every 7.22 hours the amount of bute in the horse (and the horse's bloodstream) decreases by 50%, according to Dr. Thomas Tobin, professor at the Gluck Equine Research Center, University of Kentucky. Tobin, who has performed extensive research on the metabolism of bute and other common drugs in horses, reports that with a half-life of 7.22 hours, 90% of the bute remaining in the horse will be metabolized (i.e., gone from the horse) at the end of each day. To better understand this, consider the 1.8 g of bute administered to the horse IV: In the first 24 hours, the amount of drug in the horse decreases by 90%, leaving 0.18 g or 180 mg total in the horse. This exceeds the allowable amount per ARCI guidelines. After another 24 hours, however, an additional 90% of the bute remaining in the horse is metabolized. The horse now has only 1% of the original dose left in the system: 0.018 g (18 mg). Note that this is still above the cited cut-off value mentioned above (0.015 g). However, this 0.018 g is what is present in the entire horse, including all its tissues, not just the bloodstream. The amount of bute in the bloodstream will be lower. Just like people, horses have variability in their metabolism between individuals. Not all horses metabolize bute at the same rate. The half-life in one horse might be as fast as five hours or as slow as eight or nine hours in another horse. To account for differences in horses' metabolisms, the tolerance interval was determined using a specific technique called a “95/95 tolerance.” To determine the 95/95 tolerance, the RMTC doses about 20 horses and looks at the blood levels of the medication at 48 hours post-administration. Those results can show some variability in bute levels at 48 hours because of horse-to-horse differences in the rate of bute metabolism. After statistically analyzing all of the bute blood levels, a regulatory threshold is then determined based on the actual measured levels of bute in all 20 blood samples 48 hours after administration. The idea is the threshold will take into account an average sampling of various metabolisms so the threshold is as fair as possible for a range of typical horses. For those worried about “false positives,” Scollay said they are extremely unlikely because in addition to this 95/95 tolerance calculation, testing laboratories also factor in a Measurement Uncertainty. “For a test to be classified as a violation the substance must be detected at a concentration in excess of the threshold PLUS the laboratory's Measurement Uncertainty for the method. No bute violations are called at the threshold of 0.3 mg/mL. With the Measurement Uncertainty, I would probably expect 0.4 mg/mL to be the minimum concentration reported by a laboratory,” Scollay explained. For more information, the RMTC released the webinar below: Dr. Stacey Oke is a seasoned freelance writer, veterinarian, and life-long horse lover. When not researching ways for horses to live longer, healthier lives as athletes and human companions, she practices small animal medicine in New York. A busy mom of three, Stacey also finds time for running, hiking, tap dancing, and dog agility training.