Case Report  Rapport de cas Fatal diphenhydramine poisoning in a dog John P. Buchweitz, Stephen A. Raverty, Margaret B. Johnson, Andreas F. Lehner Abstract — We report a fatal diphenhydramine poisoning of a 10-year-old, male poodle-cross dog with pre-existing conditions and suspected co-ingestion of ethanol. This case illustrates that diphenhydramine overdose can be fatal in certain circumstances and that analytical toxicology may play an important role in animal death investigations. Résumé — Empoisonnement mortel à la diphenhydramine chez un chien. Nous signalons un empoisonnement mortel à la diphenhydramine chez un caniche croisé mâle âgé de 10 ans ayant des conditions préexistantes et une co-ingestion soupçonnée d’éthanol. Ce cas illustre qu’une surdose de diphenhydramine peut être mortelle dans certaines circonstances et qu’une toxicologie analytique peut jouer un rôle important dans les enquêtes sur la mort d’animaux. (Traduit par Isabelle Vallières) Can Vet J 2014;55:1089–1092

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oisoning of animals with chemical agents is commonplace. Establishing whether the poisoning event was malicious, however, is difficult. In part, this is because of the vast number and types of chemical agents that may be employed. In addition, there is difficulty in discerning between ease of accessibility to the offending agent (i.e., in accidental poisonings) and intentional introduction of those agents to the animal’s environment (i.e., intentional poisoning). Most poisonings occur within a narrow range of chemical agents, including anticoagulant rodenticides, ethylene glycol, organophosphate and carbamate insecticides, strychnine, and caffeine and other methylxanthines (1). Accidental exposure to these agents results from unbridled roaming, indiscriminate oral evaluation of the environment, and access to human food and supplements. Intentional poisonings differ in that the poison is purposefully introduced into the animal’s environment, out of the normal context in which the substance resides. Examples include food bowls tainted with ethylene glycol and hot dogs or meat patties contaminated with restricted use pesticides or drugs. Companion animal poisonings have also been reported to involve household products and drugs for veterinary and human use (2). Medications, whether over-the-counter (OTC) or pre-

Diagnostic Center for Population and Animal Health, Toxicology Section, Michigan State University, 4125 Beaumont Road, Lansing, Michigan 48910 USA (Buchweitz, Johnson, Lehner); Animal Health Centre, British Columbia Ministry of Agriculture, 1767 Angus Campbell Road, Abbotsford, British Columbia V3G 2M3 (Raverty). Address all correspondence to Dr. John P. Buchweitz; e-mail: [email protected] Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. CVJ / VOL 55 / NOVEMBER 2014

scribed, contribute to companion animal fatalities; however, these poisoning events often result from accidental exposure due to ease of accessibility and lack of adequate supervision (3). There may be instances, though, whereby an owner symptomatically treats a pet with their own medication if they think the animal is unwell or in pain (e.g., paracetamol, ibuprofen, or naproxen), thus leading to an unintended consequence. Drugs, or other chemical agents, that are intentionally introduced to animals are commonly supplied in the context of a meal, treat, or drink to reduce texture and palatability inhibitions. Intentional intoxication has also been reported with secondhand cannabis smoke blown into the face of small animals as an adolescent prank (4,5). We report a multi-disciplinary, multiinstitutional forensic approach to the intoxication of a dog of advanced age with diphenhydramine, with examination of the underlying question of malicious intent.

Case description The body of a 10-year-old, male poodle-cross dog, weighing approximately 18 kg was submitted to the Animal Health Centre in Abbottsford, British Columbia, for necropsy. The animal was found by police in its residence lying dead adjacent to its deceased owner. The owner had died from blunt force trauma; however, there was no outward evidence of trauma to the dog. Instead, there were 3 empty bottles of sleep aid discovered at the scene. Two bottles were over-the-counter sleep aids containing the active ingredient diphenhydramine. The third bottle was prescription-based, containing the active ingredient Ran-Zopiclone. The dog was presented dead and frozen. There was moderate decomposition. The animal was well-muscled and there were abundant subcutaneous and visceral fat stores. Along the dorsolateral aspect of the torso and within the inguinal regions, fat lobules featured superficial hemorrhage. The heart was markedly enlarged with circumferential thickening of the left ventricular free wall and at the base, there was a prominent indentation. 1089

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Figure 1.  The GC/MS chromatographic trace of the deceased dog’s stomach contents (A). Note the large peak corresponding to diphenhydramine at 14.85-min retention time. The screen was qualitative but excessive accumulation of diphenhydramine could be visually appreciated on comparison to an identical analysis performed on an unrelated dog’s stomach contents (B). Peaks in common are labeled on the control dog trace and consist of commonly seen fatty acids. In addition to the large peak at 14.85-min, there were several peaks between the diphenhydramine peak and the cis-9-hexadecenoic peak at 16.0-min. These were fatty acid ethyl esters, including the 15.47-min peak as ethyl 9-hexadecenoate (9-hexadecenoic acid ethyl ester) (CAS# 054546-22-4) and the 15.68-min peak as hexadecanoic acid, ethyl ester (CAS# 000628-97-7).

The right ventricular free wall was moderately dilated and turgid. The lungs were collapsed (atelectatic) and mottled dark red to black. Moderate amounts of dark red frothy fluid filled the tracheobronchial tree. The stomach was markedly distended with partially digested dog food pellets and fetid gas, and there was a moderate amount of partially digested food within the small and large intestines. Throughout the stomach and intestines, the serosa was variegated pale to dark red. There were no other significant gross internal or external lesions. Postmortem X-rays did not reveal any indication of trauma. There was no indication of infection or inflammation in the examined tissues. Aerobic culture of select internal viscera yielded light to heavy mixed growth of Streptococcus spp. and Enterobacter cloacae, which were likely due to postmortem invasion and bacterial overgrowth rather than representing primary pathogens. Polymerase chain reaction of pooled tissues also proved negative for canine distemper virus (6) and canine parvovirus (7). 1090

Consequently, stomach contents were submitted for toxicology screening. A general organic compound screen by gas chromatography coupled with mass spectral analysis (8,9) was positive for diphenhydramine, but not Ran-Zopiclone. Diphenhydramine was semi-quantitatively estimated at 250 ppm. Additionally, fatty acid ethyl esters (FAEE) were observed suggesting the concomitant ingestion of ethanol and, presumptively, an intentional introduction of the medication to the dog in the context of feed mixed with alcohol. Blood samples to assess circulating concentrations of diphenhydramine, ethanol, and fatty acid ethyl esters were unavailable since the dog was presented in rigor mortis. No other tissue samples were made available for analysis.

Discussion Initially, the cause of death for this dog was presumptively attributed to dilated cardiomyopathy. Moreover, periglomerular fibrosis in the kidney, bridging fibrosis in the liver, coronary arteriosclerosis, and parathyroid hyperplasia were sufficiently CVJ / VOL 55 / NOVEMBER 2014

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(mice) and 500 (rats) mg/kg BW (17). Based on this information, the LDmin in this case could have been reached with as few as 4 tablets and the LD50 achieved with approximately 40 tablets. However, with pre-existing multi-systemic pathologies, a lower dose of diphenhydramine may have been sufficient to cause death. For comparison, Lagutchik et al (15) reported a male Labrador retriever dog that ingested an estimated dose of 67 mg/kg BW of diphenhydramine with no concomitant drug exposures. On admission, the serum diphenhydramine concentration was reported at 537 ng/mL and although the concentration associated with toxicity in dogs is unknown, in humans it is . 60 ng/mL (15). With treatment this dog recovered and was discharged 24 h after admission (15). The additional finding of fatty acid ethyl esters (FAEEs) provides speculative insight into the delivery of the OTC pills, and may be significant to discerning between unintentional and intentional poisoning. These esters are produced by the reaction of fatty acids with ethanol in the presence of the enzyme fatty acid ethyl ester synthase (18), which is present in almost all tissues including the gastric mucosa (19,20). In the described case, the presence of FAEEs indirectly serves as a marker for alcohol consumption. Since the stomach was markedly distended with partially digested food, it stands to reason that alcohol may have been added to the feed and medication mixture to enhance palatability and, hence, ingestion of multiple OTC pills. Alcohol interacts with a variety of medications (21,22). These interactions may be either pharmacokinetic (affecting metabolism) or pharmacodynamic (enhancing effects) (22). When combined with other medications capable of producing sedation (e.g., antihistamines), alcohol may enhance the sedative effect of those medicines (21). Hence, it could be speculated further that the consumption of alcohol with an overdose of diphenhydramine may have produced an additive, if not lethal, combination. Interpretation of toxicology results should be in the context of the forensic, crime scene, pathologic, and ancillary diagnostic studies. Simple exposure to a toxicant does not necessarily indicate that a toxicosis has occurred. With all toxic agents, a threshold generally exists below which acute signs will not develop. Technical advancements and the improving ability to measure the presence of toxins at minute levels means we will at times detect agents at levels consistent with casual sublethal exposure but not toxicosis (1). In the current case, the stomach contents were semi-quantitatively estimated to contain 250 ppm diphenhydramine. Based on the size of the dog and degree of gastric distention at the time of necropsy, the stomach volume was estimated to be 500 mL and the amount of diphenhydramine approximated at 125 mg present at the time of sampling. This would have been consistent with an overdose, and did not take into account the quantity of diphenhydramine already absorbed, circulated, and distributed to other body compartments prior to the dog’s death and discovery. For comparison, fatal cases in humans have recorded concentrations of 8.7, 12.3, 15, 554, and 705 mg diphenhydramine in the stomach content (23,24). Aside from differences established in gender, age, and weight, variations in the stomach concentration likely reflect the quantity consumed and the temporal relationship between 1091

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severe to have contributed to ante-mortem morbidity. Both OTC sleep aids discovered near the body of the dog contained 50 mg of active ingredient diphenhydramine per tablet with 20 tablets dispensed per bottle. All 40 tablets were missing, suggesting the potential for consumption of a maximum dose equivalent to 111 mg/kg body weight (BW). Based on this observation, analytical toxicology was performed and confirmed the presence of diphenhydramine in the stomach contents (Figure 1) in sufficient quantity to substantiate the suspicion of poisoning as an immediate cause of death for the dog. No tablets or drug residue were visually evident in the stomach content. Antihistamines are H1-receptor antagonists that provide symptomatic relief from allergies and may also be used as sedatives (first-generation). First-generation antihistamines may cause adverse effects because of their cholinergic activity and ability to cross the blood-brain barrier (10). Diphenhydramine is a first-generation ethanolamine-derivative antihistamine widely used in human and veterinary medicine. It is the primary active ingredient in several brand-name OTC cold and sleep aid products such as Benadryl, Nytol, and Tylenol PM. In humans, diphenhydramine is well-absorbed orally, but undergoes first-pass metabolism in the liver with only 40% to 60% of the drug reaching the systemic circulation. The pharmacokinetics of diphenhydramine have not been studied in canines (11); however, these parameters have been evaluated in humans with peak plasma concentrations occurring within 1 to 5 h and elimination half-lives varying from 2.4 to 10 h (10). A recommended therapeutic dose for diphenhydramine in dogs is 2 to 4 mg/kg BW for the treatment of allergies or mild sedation prior to stressful situations. For an 18-kg dog, this would equate to 36 to 72 mg diphenhydramine or 1 to 1.5 tablets. The margin of safety is relatively narrow within this group of antihistamines, with adverse effects occurring at or less than 1 mg/kg above therapeutic doses (12). Therefore, 2 or more tablets may have been sufficient to initiate a cascade of potentially adverse effects for this dog. However, oral doses of 1, 3, or 10 mg/kg BW have been reported to have no effect on heart rate, mean blood pressure, or electorcardiogram in otherwise healthy dogs (13). Conversely, dogs given 10 mg/kg BW of diphenhydramine in a sleep study had significantly increased drowsiness and non-REM (rapid eye movement) sleep (14). In living animals, hyperactivity, depression, hypersalivation, tachypnea, and tachycardia are the most common signs reported with ethanolamine-based antihistamines, usually occurring within 1 h of exposure (10). With overdose, dogs may also exhibit mydriasis, dry mucous membranes, disorientation, and fever (10). For animals exposed to an overdose of antihistamines, treatment with guaifenesin and supportive fluid therapy generally results in an excellent prognosis for recovery (15). The lethal dose (LD50) for diphenhydramine in dogs ranges between 24 to 30 mg/kg BW by IV administration, and death has been attributed to neuromotor excitement and convulsions, followed by respiratory failure and myocardial depression (16). The LD50 for oral diphenhydramine in dogs has not been studied; however, the range may be gleaned from extrapolated values in other species. In humans the minimum lethal dose (LDmin) is 10.1 mg/kg BW and the LD50 in rodents ranges between 114

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consumption and presentation of the decedent to the medical examiner. In the case presented, gross pathology did not provide any indication of foul play, rather necropsy and histopathology revealed degenerative conditions that may have left this dog more susceptible to the adverse effects of a drug overdose. In the absence of forensic investigations to evaluate the criminal environment and surrounding motives, malicious poisonings resulting from drug overdose in animals are difficult to prove beyond a reasonable doubt. In summary, thorough pathologic analysis and concomitant analytical toxicology provide invaluable support to a criminal investigation. The analytical evidence described herein suggests the intentional poisoning of this animal with diphenhydramine presented to it in a mixture of feed, drug, and presumably alcohol.

Acknowledgments We thank Constable D.M. Reddy from the Integrated Homicide Investigation Team of the Royal Canadian Mounted Police for his review and valuable information regarding the case history. CVJ

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Fatal diphenhydramine poisoning in a dog.

Empoisonnement mortel à la diphenhydramine chez un chien. Nous signalons un empoisonnement mortel à la diphenhydramine chez un caniche croisé mâle âgé...
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