1. “In 2004, 643 bromethalin exposures were reported to 62 major poison control centers across the United States... to our knowledge, a fatal human poisoning has never been previously reported.” Kassandra Luening and Neil Rybak

2. 21 year old male brought to emergency room after discovery with “multiple open packages of rat poison”, self reportedly ate “8 packs” Symptoms: repetitive speech, labile emotions, diminished responsiveness to direct questions, normal vital signs, one episode of emesis Treated with promethazine, thiamine, folic acid, and diphenhydramine “medically cleared” and sent to psychiatric hospital The last 2 days of 3 in psychiatric hospital, was “obtunded and catatonic”, sent to hospital for further study Stable vital signs, no fever, anisocori pupils, unresponsive to verbal or painful stimuli. Flaccid extremities Intubated and placed on a ventilator Developed distended bladder, and decreased rectal tone Declared brain dead 7 days after ingestion of “No Pest” The Case Report

3. Chemistry of MS SIM Quantification problems Lethal Dosage Process of determination of cause of death Alternate hypothesis for cause of death Overview

4. Allows for scanning of specific mass fragments, as opposed to a full scan Faster than a full scan as only a section of the spectrum is being scanned Less matrix interference The atomic mass units of the fragments of interest must be known Mass Spectrometry Selected Ion Monitoring

6. Demethylated form of bromethalin found in brain and liver samples Positive control resulted in a peak Negative control resulted in no peak

7. Knowing that there is bromethalin in the tissues does not necessarily indicate a lethal dosage, only that it was there. Problem:

8. “Because of a lack of analytical standard, no attempt was made to quantitate this analyte.” 1 The presence of bromethalin was shown, but not the quantity. The Quantification Problem

9. “accurate quantification of bromethalin in tissues is limited by the rapid photodegredation of bromethalin in sample extracts” 2 Would quantification even be accurate?

10. The lethal dosage (LD 50 ) is the amount of a substance that would kill a certain percent of a population Based on the ‘standard person’ concept and may not be suitable to all sub-populations Determining Lethal Dosage Extrapolating from human cultures Extrapolating from LD 50 of other animals Lethal Dosages

11. Cat: 0.54 mg/kg 1, 1.8 mg/kg 2 Dog: 4.7 mg/kg 2 Rabbit: 13 mg/kg 2 Guinea Pig: >1,000 mg/kg 2 Case Study Patient: 0.33 mg/kg Not quantified experimentally Based on self report of the patient who had shown diminished cognitive function, as well had a chronicled history of strange behaviour unreliable source of information Bromethalin Lethal Dosages

12. “the ischemic neuronal changes seen on microscopic examination are likely related to his terminal diagnosis and do not necessarily reflect bromethalin toxicity” 1 “the sensitivity of humans to bromethalin has not been previously studied, and the LD 50 in people is unknown” 1

13. 1.History of exposure to potentially toxic dose 2.Clinical signs 3.Physiological indicators Histologic presence of white matter vacuolization 4.Detection of drug in the body Bromethalin found in brain and liver samples It’s possible to use this same methodology to identify barbiturates as a possible cause of death Determination of Cause of Death in Case Study

14. Central Nervous System depressants Resulting in mild sedation, to total anesthesia Possible side effects include; hypotensia, ataxia, mortality, sedation (coma), areflexia, apnoea, hypotension, hypothermia Widely prescribed and frequently used for intentional self poisoning 4 15 th most common medication class associated with fatal poisoning in the US 4 “the collective experience (and comfort) of clinicians for managing patients with severe barbiturate toxicity is fading” 4 Barbiturates

15. A urine drug screen was positive for barbiturates Lethal dosage ranges from 30 µg/ml to 80 µg/ml depending on which barbiturate 5 Combined with other CNS depressants, toxicity can increase 5 Patient was treated with promethazine, and phenhydramine; both are strong sedatives 1.History of Exposure to Toxic Dose

16. Changes in alertness, decreased interpersonal function, severe confusion, areflexia, ataxia, pupil effects, decreased urine production, ventilation required, possible complications such as pneumonia, coma, brain death, mortality These signs were apparent in the case study patient Patient exhibited; repetitive speech, labile motions, diminished response to direct questioning, aniscoric pupils, flaccid extremities (ataxia), and was placed on a ventilator, coma, brain death, declared dead 2.Clinical Signs of Barbiturate Overdose Barbiturate OverdosePatient symptoms Changes in alertness Decreased interpersonal function Severe confusion Ataxia, areflexia Pupil effects Ventilation required Possible complications- pneumonia Coma Possible brain death mortality Repetitive speech Diminished response to questions Labile emotions Flaccid extremities Aniscorcic pupils Placed on ventilator Acute bronchoalveolar pneumonia Coma Brain death Death

17. Barbiturate overdose can lead to global ischemia, a complete stoppage of blood flow to the brain EEG performed on patient a day before death indicated low amplitude voltage, which is a non specific finding consistent with global ischemia and impending brain death Other possible explanations for white matter vacuolization Can be caused by PCP and ketamines Carbon tetrachloride exposure from cleaning agents can cause white matter vacuolization 6 It has been shown that bromethalin exposure at an anticipated lethal dosage does not necessarily lead to death 7 3.Physiological Indicators

18. An initial urine screening was positive for barbiturates No further testing was done “The initial positive urine barbiturate screen was never accounted for in the medical records reviewed.” 4.Detection of Barbiturate in the Body

19. Urine screening show barbiturates Indicated a history of drug use Clinical signs of barbiturate overdose were seen Global ischemia, a result of barbiturate overdose was observed in the patient MS SIM analysis was only done for expected demethylated bromethalin, no follow up was done on the positive screening in the urine analysis Barbiturate Poisoning Diagnosis

20. The bromethalin found in the patient was not quantified The LD 50 of bromethalin in humans is not known The method used to determine bromethalin poisoning as cause of death could also be used to determine barbiturate poisoning as cause of death Summary

21. It cannot be positively determined that Bromothalin poisoning was the only possible cause of death in this case study. Conclusion

22. 1.Pasquale-Style, M., Sochaski, M., Dorman, D., Krell, W., Shah, A., Schmidt, C., Fatal Bromethalin Poisoning Journal of Forensic Science 2006 vol 51, pp. 1154 – 1157 2.Dorman, D., Simon, J., Harlin, K., Buck, W., Diagnosis of Bromethalin Toxicosis in the Dog J Vet Diagn Invest 1990 pp 123 – 128 3.Cooks, R., Soltero-Rigau, E., Kruger, T., Identification of Barbiturates by Chemical Ionization and Mass Analyzed Ion Kinetic Energy Spectrometry Analytical Chemistry 1977 vol 49 pp. 435 – 442 4.Roberts, D., Buckley, N., Enhanced Elimination in Acute Barbiturate Poisoning – A Systematic Review Clinical Toxicology 2011 vol 49 pp. 2 – 12 5.American Society of Health System Pharmacists; AHFS Drug Information 2009. Bethesda, MD. (2009), p. 2578 6.USP Convention. USPDI - Drug Information for the Health Care Professional. 17th ed. Volume I. Rockville, MD: Convention, Inc., 1997. (Plus Updates)., p. 505 7.Odabasi, M., Halogenated Volatile Organic Compounds from the Use of Chlorine – Bleach – Containing Household Products Environmental Science and Technology 2008 vol 42 pp. 1445 – 1451 8.Dorman, D., Zachary, J., Buck, W., Neuropathological Findings of Bromethalin Toxicosis in the Cat Veterinary Pathology Online 1992 vol 29 pp. 139 – 144 References

23. “the sensitivity of humans to bromethalin has not been previously studied and the LD 50 in people is unknown” 1 “no attempt was made to quantitate this analyte” 1 “the ischemic neuronal changes seen on microscopic examination are likely related to his terminal diagnosis and do not necessarily reflect bromethalin toxicity” 1