Symposium on Contaminants in the Extraction Industry and Their Impact on Consumer Safety

Sponsored by ASTM Committee D37 and Solvent Direct, October 10-12, 2023 Virtual Only

This symposium will provide a forum for evaluating the current evidence and gaps regarding the presence and measurements of contaminants that pose a risk to consumer health and safety in cannabis and hemp products with a focus on evaluating the sources of contamination (including cultivation, plant uptake, extraction, processing, manufacturing, packaging, and delivery, etc.) and how they can be mitigated.

The information presented will provide a foundation for the development of new standards and specifications related to conducting risk assessments based on common sources of contamination in the Cannabis/Hemp industries, which will enable sound approaches to identify and mitigate contaminants.

Topics for this symposium include, but are not limited to regulatory limits, standards development, toxicity impact, test methods, vaping concerns and other relevant issues for the following contaminants:

• Elemental Impurities (Heavy Metals)

• Pesticides

• Microbials

• Other Contaminants/Toxins of Interest

ABSTRACTS

Preliminary Levels and Distribution of Health-Relevant Heavy Metals in Commercially Grown Cannabis and Hemp

Authors: Stephen Goldman1, Michael Van Dyke2, Katherine James2 1Kaycha Labs, Denver, CO; 2University of Colorado School of Public Health, Denver, CO

ABSTRACT SUMMARY Toxic metal or metalloid elements (often referred to generally as “heavy metals”) are associated with adverse health effects when inhaled or ingested at low-moderate levels.1,2 Smoking or vaping cannabis products can result in inhalation exposure to heavy metals from plant absorption of metals from soil, irrigation water, or fertilizers3 and, in the case of vaping, leaching of metals from the vape devices4-7. Inhalation exposure to heavy metals has been associated with an increased risk for cancer as well as neurological, renal, cardiovascular, and hepatic effects.8-11 In most states with a legal cannabis market, there are testing and contamination limits for only four heavy metals12 (lead, cadmium, arsenic, and mercury) although New York requires eight.

The potential for exposure to heavy metals from cannabis consumption is well documented.16-20 plants and flowers are hyper-accumulators that absorb metals from soil, irrigation water, or agrochemicals.21,22 This is evidenced by the use of hemp plants for phytoremediation of metal-contaminated soil.23 There is the potential for magnification of metals concentration during the processing of harvested flowers and concentration into oils.24 Heavy metals are ubiquitous in Colorado soils and groundwater14 with –special concerns for higher levels near former mining or industrial contamination sites.15

INTRODUCTION The main objective of this proposal is to systematically quantify heavy metal levels in flower products for 21 heavy metals selected based on prevalent occurrence in soils14, fertilizers3, and irrigation water15, as well as metals that may be found in the internal components of vape devices 5-7, and metals associated with adverse health effects13. These metals include silver (Ag), arsenic (As), barium (Ba), beryllium (Be), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), mercury (Hg), lithium (Li), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), antimony (Sb), selenium (Se), tin (Sn), thallium (Tl), uranium (U), vanadium (V), and tungsten (W).

We plan to characterize the occurrence and concentration of 21 heavy metals in Colorado-market cannabis and hemp samples (flower from licensed producers only). This will establish baseline levels (microgram per gram (μg/g)) of heavy metal contaminants in consumer product flowers while accounting for the growing conditions (indoor/outdoor) that may affect these levels. Our hypothesis is that the presence of higher levels of Pb, Cd, Hg, or As will be an indicator for higher levels of some of the other 17 target metals.

TEST METHOD/OVERVIEW To assess average concentrations of heavy metals in Colorado legal market cannabis, flower (n=40, 20 each from indoor and outdoor growers) and hemp flower (n=20) will be randomly selected and deidentified by Kaycha Laboratories from their typical client samples that originate from 430 licensed Colorado cannabis producers and cultivators. The flower and oil samples will be prepared according to Kaycha’s method SOP.T.30.081.CO and analyzed using the validated method SOP.T.40.081.CO on an Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) for Pd, Cd, Hg, and As and laboratory methods for the remaining 17 metals are detailed below.

Sample Analysis: For cannabis and hemp flower analysis, representative sample aliquots of 0.18-1.0 g will be digested in four milliliters (mL) of concentrated nitric acid, one mL of hydrochloric acid and two mL of deionized water for 15 minutes using microwave heating with an Anton Paar Microwave Go system. The temperature profile is specified to permit specific reactions and incorporates reaching 190 ± 10°C in approximately 15 minutes and remaining at 190 ± 10°C for 15 minutes for the completion of specific reactions. After cooling, the vessel contents may be filtered, centrifuged, or allowed to settle and then decanted, diluted to volume with water, and analyzed by ICP-MS (Shimadzu ICPMS-2030). Quality Control: Laboratory quality control (QC) and assurance (QA) methods include blanks and spikes (n=2) duplicate samples and laboratory control samples (LCS) with each batch The LCS, fortified hemp oil, is analyzed on each run as a measure of recovery. Methods used will meet AOAC SMPR as well as be audited and certified by both CDPHE and ISO- 17025 auditors. All method validations will meet ICH Q2 (R1) criteria. Elemental impurities will be reported in micrograms per gram (μg/g). Data Analysis: Analysis of variance (ANOVA) will be utilized to evaluate variations in heavy metal concentrations among the samples accounting for within-sample type variability. Heavy metal concentrations will be log-transformed since environmentally relevant metal concentrations follow a log-normal distribution. Based on the design, we can assume that the different sample groups are independent from each other. The normality and homogeneity of variances will be checked through histogram, normality test, and Levene’s test. If there is a significant difference in heavy metal concentrations across samples, multiple comparison test (Tukey’s studentized range test) will be used to identify samples with high or low heavy metal concentrations since we have equal sample size among groups. We will conduct hypothesis testing at the 0.05 level and report p-values, test statistics, means, variances and 95% confidence intervals. The data analyses will be using SAS and following standard statistical techniques. The expected outputs for this aim will be estimates of the average, 95th percentile, and maximum concentrations of 21 heavy metals in a representative sample of Colorado legal market cannabis. RESULTS/DISCUSSION Preliminary data from Colorado in 2021 has shown that 6.9% of submitted flower samples have heavy metal concentrations above the regulated limits for at least one of the four big heavy metals (unpublished data). However, there is a paucity of published research characterizing the concentrations of multiple heavy metals in market-available cannabis and hemp flowers. Our national footprint enables us to compare state datasets. Interestingly, this dataset shows very different profiles based on the geography present. For example: Colorado Data 2022: ANALYTE Arsenic Cadmium Lead Mercury Chromium New York Data 2023: ANAL YTE Arsenic Cadmium Lead Mercury Chromium Copper Nickel Antimony OCCURRENCE(%) 57.98 49.86 42.69 0.88 74.17 OCCURRENCE(%) 100.00 100.00 100.00 1.14 100.00 0.00 100.00 2.15 100.00 13.23 100.00 3.61 100.00 0.09 AVERAGE VALUE (ppm) 0.16 0.09 0.37 9.50 0.27 AVERAGE VALUE (ppm) 0.76 RANGE (ppm) 0.000 - 54.728 0.000 - 22.432 0.000 - 426.665 0.000 - 684.923 0.000 - 89.197 RANGE (ppm) 0.000 - 95.705 0.000 - 3.104 0.000 - 136.388 0.000 - 0.473 0.000 - 211.461 0.000 - 382.386 0.000 - 336.371 0.000 - 10.000 0.02

CONCLUSIONS This project takes a systematic approach to address a problem of high public health impact. We already know that cannabis can be contaminated with metals including As, Cd, Hg, and Pb that are toxic even at trace levels. We know very little about inhalation exposure the other 17 target metals that potentially contaminate cannabis products. As cannabis use grows and legalization spreads, these are critical questions that must be addressed by industry leaders and government regulators across the U.S.

ACKNOWLEDGEMENTS Portions of this study are funded through a collaboration with the University of Colorado and the Institute of Cannabis Research, CSU Pueblo. Collaborators include Dr. Mike Van Dyke, an Associate Professor at the Colorado School of Public Health and an exposure scientist with over 20 years of experience in in human health risk assessment for occupational, community, and consumer populations. Additionally, Dr. Katherine James, an environmental epidemiologist with an extensive research portfolio in metals exposure assessment and adverse health outcomes, will be included as a collaborator. Her research was one of the first to identify chronic low-moderate arsenic exposure as a risk factor for cardiovascular disease and since then has investigated the systemic impacts of other heavy metals.

REFERENCES 1. 2. Jaishankar, M., Tseten, T., Anbalagan, N., Mathew, B. B., & Beeregowda, K. N. (2014). Toxicity, mechanism and health effects of some heavy metals. Interdisciplinary toxicology, 7(2), 60–72. https://doi.org/10.2478/intox- 2014-0009 3. Mortvedt JJ (1995) Heavy metal contaminants in inorganic and organic fertilizers. Fertilizer Research 43:55– 61. 4. McDaniel, C, SR Mallampati, A Wise (2021) Metals in Cannabis Vaporizer Aerosols: Sources, Possible Mechanisms, and Exposure Profiles. Chem Res Toxicol 34(11): 2331-2342. 5. Wagner J, Chen W, Vrdoljak G (2020) Vaping cartridge heating element compositions and evidence of high temperatures. PLoS ONE 15(10): e0240613. https://doi.org/10.1371/journal.pone.0240613 6. Hess CA, Olmedo P, Navas-Acien A, Goessler W, Cohen JE, Rule AM. E-cigarettes as a source of toxic and potentially carcinogenic metals. Environ Res. 2017;152:221-225. doi:10.1016/j.envres.2016.09.026 7. Zhao D, Navas-Acien A, Ilievski V, Slavkovich V, Olmedo P, Adria-Mora B, et al. (2019) Metal concentrations in electronic cigarette aerosol: effect of opensystem and closed-system devices and power settings. Environ Res 174:125–134, PMID: 31071493, 8. Fowles, J., Barreau, T., & Wu, N. (2020). Cancer and Non-Cancer Risk Concerns from Metals in Electronic Cigarette Liquids and Aerosols. International journal of environmental research and public health, 17(6), 2146. https://doi.org/10.3390/ijerph17062146 9. Putzhammer R., Doppler C., Jakschitz T., Heinz K., Forste J., Danzl K., Messner B., Bernhard D. Vapours of US and EU market leader electronic cigarette brands and liquids are cytotoxic for human vascular endothelial cells. PLoS ONE. 2016;11:e0157337. 10. Hess C., Olmedo P., Navas-Acien A., Goessler W., Cohen J., Rule A. E-cigarettes as a source of toxic and potentially carcinogenic metals. Environ. Res. 2017;152:221–225. 11. Gaur S., Agnihotri R. Health effects of trace metals in electronic cigarette aerosols—A systematic review. Biol. Trace Elem. Res. 2019;188:295–315. 12. Valdes-Donoso P, Sumner DA, Goldstein R. Costs of cannabis testing compliance: Assessing mandatory testing in the California cannabis market. PLoS One. 2020 Apr 23;15(4):e0232041. doi: 10.1371/journal.pone.0232041. PMID: 32324781; PMCID: PMC7179872.

1. United States Pharmacopeia, (2017) USP Chapter 232, Elemental Impurities Limits, 2017revision

2. Severson RC, Tourtelot HA. (1994) Assessment of geochemical variability and a listing of data for surface

soils of the front range urban corridor, Colorado. U.S. Geological Survey Open Report 94-648. 15. Klusman RW, Edwards KW. (1976) Toxic heavy metals in ground water of a portion of the front range mineral belt. Office of Water Research and Technology U.S. Department of Interior Washington, D. C. Report OWRT Project No. A-023-COLO. 16. Zafeiraki, E.; Kasiotis, K. M.; Nisianakis, P.; Machera, K. Macro and Trace Elements in Hemp (Cannabis Sativa L.) Cultivated in Greece: Risk Assessment of Toxic Elements. Front. Chem. 2021, 9. https://doi.org/10.3389/fchem.2021.654308. 17. Ali, N.; Hadi, F.; Ali, M. Growth Stage and Molybdenum Treatment Affect Cadmium Accumulation, Cheng, LC., Lin, CJ., Liu, HJ. et al. Health risk of metal exposure via inhalation of cigarette sidestream smoke particulate matter. Environ Sci Pollut Res 26, 10835–10845 (2019). https://doi.org/10.1007/s11356-019- 04257-4

Antioxidant Defence and Chlorophyll Contents in Cannabis Sativa Plant. Chemosphere 2019, 236. https://doi.org/10.1016/j.chemosphere.2019.124360. 18. Li, S.; Zhang, C.; Wang, S.; Liu, Q.; Feng, H.; Ma, X.; Guo, J. Electrochemical Microfluidics Techniques for Heavy Metal Ion Detection. Analyst 2018, 143 (18), 4230–4246.https://doi.org/10.1039/c8an01067f. 19. Liebling, J. P.; Clarkson, N. J.; Gibbs, B. W.; Yates, A. S.; O’Sullivan, S. E. An Analysis of Over-the- Counter Cannabidiol Products in the United Kingdom. Cannabis Cannabinoid Res. 2020, can.2019.0078. https://doi.org/10.1089/can.2019.0078. 20. Wakshlag, J. J.; Cital, S.; Eaton, S. J.; Prussin, R.; Hudalla, C. <p>Cannabinoid, Terpene, and Heavy Metal Analysis of 29 Over-the-Counter Commercial Veterinary Hemp Supplements</P>. Vet. Med. Res. Reports 2020, Volume 11, 45–55. https://doi.org/10.2147/vmrr.s248712. 21. Stonehouse, GC Phytoremediation and Biofortification Potential of Cannabis Sativa L., (2019) Master’s Thesis submitted to the Department of Biology, Colorado State University. https://mountainscholar.org/bitstream/handle/10217/195371/Stonehouse_colostate_0053N_15414.pdf? sequence=1 22. Seltenrich N. Cannabis Contaminants: Regulating Solvents, Microbes, and Metals in Legal Weed. Environ Health Perspect. 2019 Aug;127(8):82001. doi: 10.1289/EHP5785. Epub 2019 Aug 20.PMID: 31430176; PMCID: PMC6791536. 23. Bengyellaa, L, M Kuddusb, P Mukherjeed, DJ Fonmbohc, and JE Kaminskia (2021) Global impact of trace non-essential heavy metal contaminants in industrial cannabis bioeconomy. Toxin Reviews https://doi.org/10.1080/15569543.2021.1992444

ASTM Metals in Cannabis Method D8469 Authors: L. Craig Jones, Jenny Nelson; Agilent Technologies, Inc., Santa Clara, CA

ABSTRACT SUMMARY It is of utmost importance to establish consistent procedures for testing heavy metals in cannabis items. The utilization of ICP-MS is especially critical in achieving precise and sensitive analysis. This technique allows for the detection of multiple elements simultaneously, proving its value in examining complex matrices like cannabis. However, the accuracy of test results is dependent on the correct sample preparation approach, such as microwave-assisted acid digestion, which effectively extracts heavy metals from the matrix. Certified reference materials and spiked samples should also be employed to validate the analytical method for added assurance. Utilizing standardized methods and ICP-MS for heavy metal analysis in cannabis products has numerous benefits, including promoting industry transparency and accountability, ensuring uniform quality, and instilling consumer confidence. Ultimately, these methods are crucial in safeguarding public health, fulfilling regulatory obligations, and enhancing trust in the safety and quality of cannabis products.

INTRODUCTION A new standard test method for analyzing various elements in cannabis matrices was announced by ASTM International's cannabis committee (D37) in October 2022. This technique uses microwave acid digestion and ICP-MS to detect the presence of twenty-three elements, including four priority metals (As, Cd, Pb, and Hg). The published standards by the committee are highly relevant to the cannabis industry, regulators, and product consumers. The testing method can be applied to a range of cannabis-related products, including dried plant materials, concentrates, oils, extracts, and tinctures.

TEST METHOD/OVERVIEW

Instrumentation The 7850 ICP-MS was utilized for the analysis, which comes with the UHMI system and ORS4 collision/reaction cell. The Agilent SPS 4 autosampler was used to introduce the samples. The standard sample introduction system, which includes a MicroMist glass concentric nebulizer, quartz spray chamber and a quartz torch with 2.5mm id injector, was configured for the 7850 ICP-MS. Additionally, the interface was made up of a nickel-plated copper sampling cone and a nickel skimmer cone.

Sample preparation To analyze the samples, we utilized the digestion procedure outlined in the ASTM method. Homogenization is a crucial step for microwave digestion for solid samples, while non-solid ones require no further preparation. Each hemp sample weighing approximately 0.5 g was weighed into a 110 mL CEM Xpress Plus vessel. Next, we added 9 mL of HNO3 and 1 mL of HCl to each vessel. We employed the CEM MARS 6 batch microwave system to execute the digestion process and followed the heating program specified in Table 2. Upon completion of the digestion process, we diluted the samples with de-ionized water until a final weight of 100 g was achieved.

RESULTS/DISCUSSION During this presentation, we will explore the capabilities of ICP-MS in detecting heavy metals in cannabis. This method exceeds the limits set for metal concentration in cannabis and its related products. To guarantee the accuracy of the ICP-MS analysis, several laboratories conducted a triplicate analysis of three plant samples to ensure the effectiveness of the sample digestion

process. The results showed consistency with the certified concentrations from NIST, confirming the method's precision. CONCLUSIONS The ASTM method for the ICP-MS determination of As, Cd, Hg, and Pb and additional optional elements in cannabis and related products enables manufacturers to routinely test for these metals in ingredients and final products. All samples were prepared using microwave digestion in HNO3 and HCl to ensure the chemical stability of all required and additional elements and method development for the ASTM method completed by ICP-MS.

ACKNOWLEDGEMENTS Jenny Nelson, Craig Jones, Agilent Technologies, Inc. Sam Heckle, Leanne Anderson, CEM Corporation, USA REFERENCES 1. ASTM D8469-22, Standard Test Method for Analysis of Multiple Elements in Cannabis Matrices by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Oct 26, 2022, accessed May 2023, https://www.astm.org/d8469-22.html 2. Committee D37 on Cannabis, accessed May 2023, https://www.astm.org/get- involved/technical-committees/committee-d37 3. Agilent IntelliQuant for ICP-MS, Fast, automated semiquantitative ICP-MS analysis provides greater sample insight and confidence in the results, Agilent publication, 5994- 2796EN 4. Tetsuo Kubota, Simplifying Correction of Doubly Charged Ion Interferences with Agilent ICP-MS MassHunter, Fast, automated M2+ correction routine improves data accuracy for Zn, As, and Se, Agilent publication, 5994-1435EN 5. Barber CA, Bryan Sallee CE, Burdette CQ, Kotoski SP, Phillips MM, Wilson WB, Wood LJ (2022) Cannabis Quality Assurance Program: Exercise 2 Toxic Elements Final Report. (National Institute of Standards and Technology, Gaithersburg, MD), NIST Interagency/Internal Report (IR) NIST IR 8452. https://doi.org/10.6028/NIST.IR.8452

The Elemental Composition of Hemp Flower: Sources of Elemental Impurities and Implications for Consumer Product Safety Authors: Derek D. Wright, Hannah Clause, Benjamin Southwell, Mark Zierden; Lake Superior State University, Sault Sainte Marie, MI ABSTRACT SUMMARY The rapid growth of the hemp extract market has led to increasing focus on the consumer product safety of the myriad of new products entering commerce. The cannabis plant is widely accepted to be an efficient accumulator of potentially toxic elements, and its resinous nature makes it well suited to accumulate surface contaminants. Unfortunately, limited data is currently available on the occurrence of many elements in consumer cannabis, and even fewer studies have examined surface adhered particulate matter. As part of this study, 25 elements (Ag, Al, As, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, Pb, Se, Th, Tl, U, V, & Zn) were quantified in hemp produced for CBD, as well as commercially available hemp marketed for smoking. Additionally, surface adhered particulate matter was examined as a potential contributor of elemental impurities. Results confirm that hemp entering commerce in the United States contains a variety of elemental impurities, and frequently contains adhered particulate matter such as soil minerals, air pollution, agricultural additives, microplastics/textile fibers, and materials from harvesting/processing equipment. Consumer exposure potential for some elements was found to be high enough to warrant additional investigation as to the possible health effects and may justify additional oversight from regulators.

INTRODUCTION The legalization of industrial hemp (cannabis containing <0.3% THC by weight) in the United States by the 2018 Farm Bill has led to the increasing popularity and consumer use of hemp derived products focused on non-psychoactive cannabinoids such as cannabidiol (CBD). The rapid proliferation and often unclear legal status of consumer CBD products has created a significant challenge for public health and regulatory agencies, which has been compounded by the rapid expansion of state regulated recreational and medical marijuana programs despite the continued marijuana federal Schedule I status. This has led to an inconsistent patchwork of state level regulations based on limited scientific evidence, and the potential for unsafe consumer products to enter the marketplace. One group of contaminants in cannabis products is of concern is the presence of potentially toxic elements, particularly in cannabis products intended for inhalation. While the availability of published studies on elemental accumulation by cannabis plants is still somewhat limited, a general consensus has emerged that cannabis is both tolerant of elevated heavy metals in soils, and is an efficient accumulator of several potentially toxic elements such that it is a promising candidate for phytoremediation applications. There remains however, little publicly available data on the elemental composition of cannabis for many elements of interest. Additionally, the resinous nature of cannabis leads to a significant potential to accumulate surface contaminants. This may further increase consumer exposure, but there is almost no literature documenting this potential route of accumulation. Here we present data on the elemental composition (Ag, Al, As, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, Pb, Sb, Se, Th, Tl, U, V, & Zn) and particulate contaminants present in hemp intended for CBD production from the HEMP-TRIM in Michigan (outdoor research fields in Chatham, Bay Mills, and Harbor Springs), as well as from commercially available consumer hemp sources marketed for consumption by smoking.

TEST METHOD/OVERVIEW The elemental composition of hemp flower was quantified by inductively coupled plasma-mass spectrometry (ICP-MS). Dry flower was homogenized with a metal free grinder (Distek Inc.) and microwave digested with HNO3/HCl (CEM Mars 6). ICP-MS analysis was performed with an Agilent 7800 and 4x aerosol dilution using EPA 6020, modified. Examination by stereomicroscopy was performed with either a Nikon SMZ1000N or an Olympus SZH-10 equipped with an apochromatic common main objective. Intact flower (1-3 g) was examined with a thorough screening of the whole flower surface at 20x magnification, and then at higher magnification, up to 80x, when potential foreign material was identified. Typical examination time for each sample was ~30-60 min. Analysis of particulate material was performed by scanning electron microscopy with energy dispersive x-ray spectroscopy (SEM-EDS) or laser induced breakdown spectroscopy (LIBS). SEM-EDS analysis was performed on intact uncoated flower samples under low vacuum conditions with an acceleration voltage of 15kV. LIBS data was collected on intact flowers using a UV laser with automated elemental identification performed using three emission lines per element. Exposure potentials were calculated based on ICP-MS results using a maximum exposure model (i.e., the total elemental content was assumed to be potentially available for inhalation) due to the limitations of available data on actual elemental exposure for a broad suite of elements. RESULTS/DISCUSSION The median and maximum dry weight elemental composition of hemp samples from this study are presented in Table 1. For the majority of elements, the maximum concentrations observed are within a factor or 2-3 of the median concentration. Sodium (15x), Chromium (6x), Silver (5x) and Cd (8x) however show a greater range, though it is unclear the degree to which this reflects genotypic/phenotypic variability in the plants, environmental conditions, or potential contamination from processing, etc. Table 1: Elemental Composition of Hemp A wide variety of surface contaminants were observed (Fig. 1) including soil particles, fertilizers/soil amendments, and synthetic polymers. Additional particulate contamination (data not shown) included stainless steel particles (likely harvesting/processing equipment) with AgCl surface contamination on the particle, as well as fly ash (spherical silicates). While these observations are qualitative in nature, most of the commercially available hemp we examined had at least one or more textile fiber/probable microplastic, with some containing many fibers. The degree to which these may pose a human health risk independent of their contribution to elemental contamination is uncertain, but it illustrates the importance of further studies of foreign matter contamination and the development of standardized methodologies.

Figure 1: Particulate contaminants observed in hemp samples. Additionally, we compared the median and maximum values of elemental composition in our data set to dose based reference values from USP 232 and ICH Q3D to estimate exposure potential. While there is consensus for regulating the “Big Four” elements (As, Cd, Hg, & Pb), our data suggests that a broader element suite should be evaluated for human health risk. In particular, it appears that the Cannabis plant frequently accumulates a significant quantity of Cu, and may accumulate relevant quantities of several other elements (Cr, V, Mo, etc.) that are not frequently regulated in state cannabis compliance programs. Table 2: Exposure Potential based on consumption of 5 g d-1 or 2 g d-1 of hemp by inhalation (smoking).

CONCLUSIONS

1. 1) Cannabis may bioaccumulate significant quantities of many elements.

2. 2) Cannabis may contain sufficient quantities of some elements (i.e., Cu) to pose a potential human exposure risk, including elements not commonly regulated by state cannabis compliance programs.

3. 3) Cannabis can accumulate external material (foreign matter) such as: soil minerals, air pollution, agricultural additives, microplastics/textile fibers, and materials from harvesting/processing equipment.

ACKNOWLEDGEMENTS Funding for this work was provided by USDA-NIFA Award: 2020-38424-31819, NSF Award: 2215270, and the Lake Superior State University College of Science & Environment. Special thanks to Steven Yanni (Bay Mills Indian Community) and James DeDecker (Michigan State University Upper Peninsula Agricultural Extension) for providing samples. A portion of the SEM-EDS data was collected by David Edwards (JEOL USA) and the LIBS data was collected by Andrew Harvey (Keyence Corporation). ICP-MS analysis and microscopy was performed by LSSU students Carmen Kincaid, Emily Hebert, and Ashley Render.

REFERENCES 1. Louis Bengyella, Mohammed Kuddus, Piyali Mukherjee, Dobgima J. Fonmboh & John E. Kaminski (2021): Global impact of trace non-essential heavy metal contaminants in industrial cannabis bioeconomy, Toxin Reviews, DOI: 10.1080/15569543.2021.1992444 2. Benjamin Southwell and Derek Wright. Keeping it clean: Contamination control in the cannabis industry. 3. David Edwards and Hannah Clause. Using SEM for Foreign Matter Identification. Presented at: Confidence in Your Cannabis: Exploring New Horizons

Comparison of State-Level Regulations for Cannabis Contaminants and Implications for Public Health Authors: Maxwell C. K. Leung; School of Mathematical and Natural Sciences, Arizona State University, Glendale, AZ ABSTRACT SUMMARY The presence of contaminants in cannabis presents a potential health hazard to recreational users and susceptible patients with medical conditions. Due to the federally illegal status of cannabis, there are no unified regulatory guidelines mitigating the public health risk of cannabis contaminants in the U.S. Here, we examined the current landscape of state-level contaminant regulations, cannabis contaminants of concern, as well as patient populations susceptible to contaminants. As of May 18, 2022, 36 states and the District of Columbia listed a total of 679 cannabis contaminants as regulated in medical or recreational cannabis, including 551 pesticides, 74 solvents, 12 elemental impurities, 21 microbes, five mycotoxins, and 16 other contaminants. Different jurisdictions showed significant variations in regulated contaminants and action levels ranging up to four orders of magnitude. The compliance testing records of 5,654 cured flower and 3,760 extract samples – which accounted for approximately 6% of California’s legal cannabis production in 2020 - 2021 – showed a failure rate of 2.3% was identified for flowers and 9.2% for extracts. Insecticides and fungicides were the most prevalent categories of detected contaminants, with boscalid and chlorpyrifos being the most common. The contaminant concentrations fell below the regulatory action levels in many legalized jurisdictions, indicating a higher risk of contaminant exposure. While individual jurisdictions can implement their policies and regulations for legalized cannabis, this study demonstrates the urgent need to mitigate the public health risk of cannabis contamination by introducing national-level guidelines based on conventional risk assessment methodologies and knowledge of patients’ susceptibility in medical use. INTRODUCTION The broader use and state-level legalization of cannabis have gained significant public interest and support in recent years. Approximately half of U.S. adults reported that they have used cannabis at least once in their lives, with nearly 55 million reported using cannabis within the past year. In 2017, the U.S. legal cannabis market was valued at $10 billion. It is projected to be worth $50 billion in 2026. While public interest focuses mainly on the socioeconomic impact and medical benefits of legalized cannabis, little attention has been paid to its implications in chemical exposure and consumer safety. Like other agricultural commodities, cannabis is prone to contamination by pesticides, elemental impurities, microbes, and mycotoxins. Additionally, solvent residues are an increasing concern for cannabis extracts. The presence of these contaminants presents a potential health hazard not only to regular cannabis users and the general public but also to people with specific health conditions that make them susceptible to harmful contaminants. Immunocompromised patients with cancer and HIV, women of reproductive age, and patients with seizures and epilepsy are among those who are susceptible to the health hazards of pesticide and microbial contaminants. At the federal level, cannabis is still listed as an illegal Schedule I substance. This limits the efforts of several federal agencies in assessing and mitigating the public health risk of cannabis contamination in the U.S. Individual state-level jurisdictions where cannabis has been legalized are currently implementing their policies and regulations for cannabis contaminants. This is similar to the state and local policies concerning facemasks and vaccination mandates to prevent the spread of COVID-19, where inconsistent policies on a jurisdiction-by-jurisdiction basis have resulted in poor public health outcomes in certain jurisdictions.

TEST METHOD/OVERVIEW This study aims to examine the current landscape of state-level contaminant regulations for cannabis. First, we examined the regulatory documents for medical and recreational cannabis in all legalized U.S. jurisdictions and compiled a complete list of regulated contaminants, namely pesticides, elemental impurities, solvents, microbes, and mycotoxins. Second, we data-mined the compliance testing records of 5,654 cured flower and 3,760 extract samples that accounted for approximately 6% of California’s legal cannabis production in 2020 - 2021. Lastly, we discuss the susceptibility of patients to harmful contaminants in medical cannabis RESULTS/DISCUSSION We began by surveying the contaminant regulation of drug-type Cannabis sativa L. in 50 states and Washington, D.C. As of May 18, 2022, 36 states and Washington, D.C. had legalized medical cannabis, while 17 states and Washington, D.C. had legalized both medical and recreational cannabis. Thirty-one states and Washington, D.C. listed a total of 679 cannabis contaminants in their regulatory documents. However, only 23 jurisdictions listed specific contaminants in all four categories (i.e., pesticides, solvents, elemental impurities, and microbes/mycotoxins) in their regulations. Five jurisdictions did not mention any specific contaminants and one jurisdiction provided no action levels for its list of contaminants. Shortcomings of contaminant regulations by categories. We systematically examined the 551 pesticides, 74 solvents, 12 elemental impurities, 26 microbes and mycotoxinslisted by the 31 states and Washington, D.C. in their regulatory documents (Figure 1). Pesticides, the largest category of regulated contaminants, included 174 insecticides, 160 herbicides, 123 fungicides, and 17 miticides (i.e. acaricides). The miticides bifenazate and etoxazole, the fungicide myclobutanil, and the insecticide imidacloprid were regulated by the highest number of jurisdictions (27). There was a large variation in regulated pesticides and action levels in different jurisdictions. Twenty-eight jurisdictions listed elemental impurities – often categorized as “metals” or “heavy metals” – in their regulatory documents. Arsenic, cadmium, lead, and mercury were listed by all 28 jurisdictions with action levels ranging from 0.00009 to 10 ppm. Other regulated elemental impurities included chromium (listed by six jurisdictions), nickel (two jurisdictions), and copper (two jurisdictions). The most common regulated solvents were hexane (25 jurisdictions), heptane (25 jurisdictions), butane (24 jurisdictions), toluene (24 jurisdictions), and benzene (23 jurisdictions). Action levels for solvents varied widely from Figure 1. Histograms showing the number of listed cannabis contaminants regulated in each of the 36 legalized states and Washington, D.C.’s regulatory documents for cannabis as of May 18, 2022. The four main categories of contamination listed are presented as separate panels. Five of the states named no individual contaminants for any category.

zero tolerance to 5000 ppm. This is consistent with the fact that some solvents naturally occur in in Cannabis plants (e.g., ethanol), while others are processing residues that present in a lesser amount (e.g., butane). Thirty-six jurisdictions mentioned microbial contamination in their regulatory documents, but only 32 listed specific microbes and mycotoxins. The most common regulated contaminants in this category included E. coli and/or Shiga toxin-producing E. coli (listed by 31 jurisdictions), Salmonella (26 jurisdictions), aflatoxin B1, B2, G1, and G2 (27 jurisdictions), and ochratoxin A (26 jurisdictions). Fusarium mycotoxins – which were prevalent contaminants in several agricultural commodities – were not regulated by any jurisdictions. Contaminant load in California cannabis samples. We data-mined the compliance testing records of 5,654 flower and 3,760 extract samples in California and identified 141 flower and 423 extract samples containing a detectable level of regulated contaminants. Of the contaminated samples, 132 flower and 347 extract samples failed the state’s regulatory action levels. The flower samples contained 39 of the 103 regulated contaminants in California and the extract samples contained 61 of the regulated contaminants. Overall, we calculated a total failure rate of 5.1% for all cannabis samples, including a 2.3% failure rate for flowers and a 9.2% failure rate for extracts. Our result was consistent with the cumulative failure rate of 4.2% in all certified cannabis analyses in California’s legal market since the inception of the California Department of Cannabis Control in 2017 (i.e., 8,476 failed samples due to contamination or other issues in a total of 204,163 tested samples). The failure rates are also consistent with the reported rates from other legal markets in the U.S. including Colorado and Oregon. Organophosphate and pyrethroid pesticides were the two most commonly found classes of pesticides in cannabis (Figure 2). We also identified a number of legacy pesticides and some that were being phased out, including chlordane (banned by the U.S. EPA in 1988) and chlorpyrifos (banned by California EPA in 2020), indicating ongoing use of theoretically banned pesticides. Susceptibility of medic patients to cannabis contaminants. For three reasons, our results indicate potential health risk to patients with susceptible medical conditions. First, the illegal market of cannabis is still thriving. Cannabis that fails compliance testing is likely sold on the illegal market. Pesticides are sometimes added to illegal cannabis as adulterants (i.e., “spikes”) in high quantities. In one case, the herbicide paraquat was found at a dry weight ratio of 2.5%. Second, patients are often given no information by their medical providers regarding the safety issues of cannabis use. We conducted a preliminary study and surveyed 20 neurologists and movement disorder specialists who treat primarily individuals with Parkinson’s disease. Most of them are unaware of any commonly found contaminants or thought there weren’t any present at all. Third, the action levels are set up by individual states without federal guidance and information on

pesticide exposure and mixture toxicity. The action level for one pesticide may vary up to four orders of magnitude. For instance, the action levels of dichlorvos and dimethoate in cannabis ranged from zero tolerance in California to 1 ppm in several states (Figure 2). In contrast, the U.S. EPA tolerances of dichlorvos and dimethoate range from 0.5 to 2 ppm in most fruits and vegetables. The lack of unified regulatory guidelines can discourage compliance of cannabis manufacturers. It also subjects patients to a higher level of contaminant exposure in some states. CONCLUSION The increasing use of cannabis for medical purposes, together with the lack of functional regulatory oversight, makes it a potential route of harmful contaminant exposure to susceptible populations. This study demonstrates the urgent need to mitigate the public health risk of cannabis contamination by introducing national-level guidelines based on conventional risk assessment methodologies and knowledge of patients’ susceptibility in medical use. ACKNOWLEDGMENTS I would like to thank all my students and collaborators who contributed to this work (see References), as well as the funding support of the ASU Presidential Scholarship. REFERENCES Jameson et al., 2022. Environmental Health Perspectives 130: 097001. Pinkhasova et al., 2021. Current Research in Toxicology 2: 140-148. Leung et al., 2019. Reproductive Toxicology 85: 12-18.

Rapid, Efficient and Safe Sample Preparation of Mixed Cannabis Matrices for Analysis of Multiple Trace Elements Authors: Alicia D Stell, Samule Heckle, Macy Harris; CEM Corporation, Matthews, NC ABSTRACT SUMMARY Certain elements can cause adverse effects on human health. Particularly toxic heavy metals such as arsenic, cadmium, lead, and mercury are persistent once released into the environment and can accumulate in cannabis plants. As more is learned about cannabis and its products, more elements beyond just the four main toxic heavy metals are of interested. Cannabis-based products such as foods, oils, tinctures and more should be tested for the presence of multiple elements to ensure consumer safety and product quality. Cannabis-infused products have grown in popularity and must adhere to increased regulation, necessitating the need for rapid and accurate trace metals analysis. Microwave-assisted acid digestion was used to completely and safely digest a batch of 24 mixed matrices in as little as 35 minutes. Following ASTM D8469 excellent recovery for 23 metals was achieved for a wide variety of cannabis matrices including SRMs. In the presentation the sample preparation of these samples is discussed, including tips for best results. This approach provides a rapid, efficient and safe process for trace metals analysis. INTRODUCTION In 1970, marijuana was designated a Schedule 1 drug, under the Controlled Substances Act, making it nearly impossible for laboratories to perform cannabis research. However, medicinal use of cannabis is now legal in Canada and many US states. With the passage of the Farm Bill in 2018, it is now federally legal to grow and process hemp in all 50 states. All of this interest in medical cannabis has highlighted the need for good analysis methodology in this relatively young market. Cannabis analysis is still developing standardized protocols, requirements, and acceptable testing practices. Typical testing requirements for cannabis and its products include heavy metals analysis, pesticide residue, and the potency of active ingredients such as tetrahydrocannabinol (THC). Herein a rapid, efficient, and safe microwave-assisted acid digestion of mixed cannabis matrices for trace metals analysis following ASTM D8469-22 will be discussed. TEST METHOD/OVERVIEW A batch microwave digestion system, equipped to run 25 samples of 110 mL volume vessels, was used to digest the varied cannabis matrix types. All samples required special preparation, based on the qualities of each sample, in order to obtain a homogeneous 0.5 g approximate sample weight deposited on the bottom of the vessel. Nine mL of HNO3 and 1 mL HCl were added to each vessel and the samples were allowed to predigest for 15 minutes, prior to sealing and placing the vessel in the turntable. Samples were then microwave digested at a temperature of 210 °C, achieved in less than 35 minutes. Both spiked and SRM samples were digested and analyzed. Upon successful digestion, all samples were diluted to 50 mL with deionized water for ICP-MS analysis. RESULTS/DISCUSSION This batch microwave-assisted acid digestion method was able to successfully digest all samples in a mixed batch. All of the samples were completely digested, yielding clear and particulate- free solutions upon dilution with deionized water. Excellent recoveries within requirements were achieved for all 23 elements analyzed. This an ideal process for the cannabis industry because it is able to successfully and safely digest batches of mixed matrices, including foods, oils, tinctures, creams, and plant materials, in as little as 35 minutes.

CONCLUSIONS A batch microwave digestion system was able to digest a wide variety of cannabis and hemp samples in mixed batches producing digestate that was suitable for analysis. Analysis of all samples showed excellent spike recovery and gave great confidence in the actual levels of trace metals in the SRM samples. ACKNOWLEDGEMENTS We would like to thank Agilent for their expertise and assistance with the analysis. REFERENCES ASTM D8469-22

Understanding Sources of Elemental Contaminants in Cannabis and Hemp: Benefits of a Risk Assessment Strategy Authors: Robert Thomas; Scientific Solutions, Gaithersburg, MD ABSTRACT SUMMARY The pharmaceutical industry took over 20 years to change from a semi-quantitative method to monitor a small group of heavy metals to finally arrive at regulations for 24 elemental impurities in drug products, listed by their permitted daily exposure (PDE) limits and categorized by toxicological impact and method of administration (oral, parenteral, inhalation, transdermal). The entire premise was based on carrying out a comprehensive risk assessment study of the elements’ toxicity and the likelihood of finding them somewhere in the drug manufacturing process, which was fully documented in ICH Q3D guidelines for elemental impurities. The cannabis industry cannot move beyond testing just four heavy metals until this type of risk assessment study is carried out. The objective of this presentation is to offer guidance as to which elemental contaminants are worthy of consideration, based on likely sources derived from the cultivation, extraction, processing, packaging and delivery of cannabis and hemp consumer products and to explore how this well-established pharmaceutical risk assessment process could be adapted by the cannabis industry. The presentation will focus on four major areas:

• The pharmaceutical risk assessment approach

• Can risk analysis be adapted for the cannabis industry?

• Sources of elemental contaminants derived from cultivation practices

• Contributions from the cannabis extraction and manufacturing process INTRODUCTION The lack of federal oversight with regard to heavy metals in medicinal and consumer cannabis products in the US has left individual states to regulate its use. Cannabis is legal in 37 states, while 18 states and Washington, DC, allow its use for adult recreational consumption (1). However, the cannabis plant is known to be a hyper-accumulator of heavy metals in the soil, so it is critical to monitor levels of elemental contaminants to ensure cannabis products are safe to use. Unfortunately, there are many inconsistencies with heavy metal limits in different states where cannabis is legal. The vast majority of these states define the “big four” heavy metals: lead (Pb), arsenic (As), cadmium (Cd), and mercury (Hg). New York State also requires the testing for chromium (Cr), nickel (Ni), copper (Cu), antimony (Sb) and zinc (Zn), while Michigan requires inorganic As (not total As) and also adds Cr, Ni, and Cu. Maryland and a few other states also include Cr as well as the big four. Some states base their limits directly in the cannabis, while others are related to human consumption per day. Others take into consideration the body weight of the consumer, while some states do not even have heavy metal limits. To complicate the situation, certain states only require heavy metals in the cannabis plant/flower, while some give different limits for the delivery method such as oral, inhalation, or transdermal (2). These inconsistencies and the fractured nature of state-based limits would make it extremely complicated to monitor at the federal level, because currently all regulations apply only in the state where the cannabis is grown, processed, and sold. And since the federal government still considers cannabis a Schedule I drug there should be no interstate commerce with regard to cannabis products. However, it is now legal to grow hemp anywhere in the US for the production of CBD-based consumer products, so it will be interesting to see how the Department of Agriculture regulates

the hemp industry at the federal level, when cannabis is regulated by the individual states where it is legal. OVERVIEW So clearly there is a need for more consistency across state lines, particularly as the industry inevitably moves in the direction of federal oversight. This is further compounded by the fact that there is compelling evidence in the public domain that only monitoring the big four heavy metals is totally inadequate to ensure consumer safety. But how many metals should there be in an expanded list? At a recent ASTM workshop dedicated to the measurement of heavy metals in cannabis and hemp consumer products, compelling evidence was presented by a number of researchers that suggested 10-15 elemental contaminants are worthy of consideration (3). Moreover, the National Institute of Standards and Technology (NIST) is developing a 13-toxic element hemp certified reference material (CRM) through its CannaQAP Program to include Pb, Cd, As, Hg, beryllium (Be), cobalt (Co), Cr, manganese (Mn), molybdenum (Mo), Ni, selenium (Se), uranium (U), and vanadium (V) (4). In addition, ASTM International's D37 Committee is in the process of developing a standardized ICP-MS method for up to 23 different elemental contaminants in cannabis (method in review). So, what will be a realistic panel of botanical heavy metals to monitor in cannabis consumer products, particularly as there is no comprehensive understanding of the sources of elemental contaminants in the cannabinoid production processes. Moreover, besides the big four, there is no consensus on the toxicity impact of other heavy metals in cannabis and hemp, as there has been no risk assessment study carried out with regard to heavy metal contaminants and for that reason, consumer safety is likely being compromised. The only point of reference we have at this current time for what could be a federally regulated panel is the FDA approved CBD-based drug Epidiolex, which is available in the US to treat childhood seizures. Manufactured by UK-based GW Pharmaceuticals, it went through the regulatory process four years ago to get it approved in the US and had to show compliance by meeting permitted daily exposure (PDE) limits for up to 24 elemental impurities as defined in USP Chapter 232 and ICH Q3D guidelines. In fact, it’s worth pointing out that the USP recently published for public comment in its pharmacopeial forum (PF 48-1), a draft monograph for CBD intended for use as an API for drug formulations, which stated that: “Elemental impurities in official drug products are controlled according to the principles defined and requirements specified in Elemental Impurities—Limits 232, as presented in the General Notices 5.60.30.” In the long term, this could possibly indicate that the FDA will regulate CBD products for up to 24 elemental contaminants when it eventually has oversight of the cannabis industry. But more importantly, in the short term it implies that CBD being manufactured in the US for recreational or medicinal purposes is not pure enough for federally-approved drugs, because it only has to comply with the state’s maximum limits for heavy metal contaminants, which in most US states is typically only Pb, Cd, As, and Hg. However, it’s important to stress that a panel generated by pharmaceutical regulators isn’t necessarily one that should be used by the cannabis industry, as the process for manufacturing cannabinoids is very different to drug products. So, the objective of this white paper is not to provide evidence as to what elemental contaminants the industry should be monitoring, but to offer guidance on which ones are worthy of consideration by implementing a comprehensive risk assessment study supported by evidence from information in the public domain and other sources about what metals are likely candidates. DISCUSSION The cannabis industry can learn a great deal from the pharmaceutical industry, as it went through this process over 20 years ago when it updated its 100-year-old semi-quantitative sulfide

precipitation colorimetric test for a small suite of heavy metals to eventually arrive at a list of 24 elemental impurities in drug products defined as permitted daily exposure (PDE) limits and classified by their toxicity and the probability of inclusion in the drug product. These procedures were described in United States Pharmacopeai Chapters 232 - Elemental Impurities (5) and 233 – Procedures (6) together with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q3D guidelines on elemental impurities (7). Note: The ICH is an international consortium of pharmaceutical regulatory authorities and pharmaceutical industry groups to discuss scientific and technical aspects of pharmaceuticals and develop guidelines. These new guidelines defined maximum PDE limits based on well-established elemental toxicological data for drug delivery methods (including oral, parenteral, inhalation, and transdermal), together with the analytical methodology using techniques such as plasma spectrochemistry to carry out the analysis. This meant that pharmaceutical manufacturers were required to not only understand the many potential sources of heavy metals in raw materials and active ingredients, but also to know how the manufacturing process contributed to the elemental impurities in the final drug products. The beginning of the journey to regulate elemental impurities in pharmaceuticals in the late 1990s can be compared to the production of cannabis and hemp derived products today, where the sources of elemental contaminants, the toxicological impacts of the contaminants and the methodology to perform the measurements were not fully understood or developed. In particular, the elemental toxicological guidelines to regulate the cannabis industry are being taken very loosely from a combination of methods and limits derived by the pharmaceutical, dietary supplements, food, environmental, and cosmetics industries. Even though the process of manufacturing cannabinoids might be similar in some cases to drugs and herbal medicines derived from natural products, the consumers of cannabis and hemp products are using them very differently and in very different quantities, particularly compared to pharmaceuticals, which typically have a maximum daily dosage. The bottom line is that heavy metal toxicological data generated for pharmaceuticals over a number of decades cannot simply be transferred to cannabis, hemp, and their multitude of products. So, let’s take a detailed look at how the pharmaceutical industry approached bringing in comprehensive regulations using a risk assessment approach of those elements that are not only known to be toxic but also likely to be potentially found somewhere in the drug manufacturing process. CONCLUSION It took the pharmaceutical industry over 20 years to fully-understand all the sources of elemental impurities in the manufacture of drug products. The sector finally accomplished this by classifying the impurities’ toxicity impact and the risks of finding these impurities throughout the entire manufacturing process. It is clear that the cannabis industry has a great deal to learn about this process. The way to minimize heavy metals in cannabis and cannabis products is to first understand and characterize the cultivation process. Unfortunately, this is often very challenging, particularly if the plants are being grown outdoors. However, by carefully selecting the right cultivars, understanding the soil chemistry and the use of high purity fertilizers, nutrients, and water, the industry can minimize the plants’ uptake of elemental contaminants. Moreover, by optimizing the extraction and purification method, processors have the ability to reduce levels of heavy metals in the final cannabinoid extracts. Very often there are many choices when selecting an extraction technology, depending on the desired extract or the products being made for a therapeutic outcome. It is clear that elemental contaminants can be minimized by optimizing the entire production process, including extraction solvent,

temperature/pressure/flow conditions, and the other purification techniques, including evaporation, distillation, and filtration. However, this must also be extended to include the packaging and delivery systems, which are all important areas to characterize in order to ensure that cannabis consumer products are free of heavy metals and safe for human consumption. A final word of caution! The insatiable consumer appetite for cannabis products in the US is being fulfilled from outside the country. Yunnan Province in southern China is now producing CBD-products for the US market (8). This should not be surprising, considering the US industry cannot produce enough to supply the huge demand. What is more disturbing is that the metal refining for the electronics industry in China has produced some of the most contaminated waste sites in the world (9). Experience has cautioned us that consumer products coming from China are not always of the highest quality. So, it is imperative that no matter where the products are sourced, especially if it is from outside the US, testing the hemp and CBD products for a comprehensive suite of elemental contaminants is critically important. Our appetite for the cannabis and hemp plant and their medicinal and recreational properties is unlikely to diminish in the near future, so we are going to have to balance that with its natural instinct to absorb heavy metal from its growing medium. Hopefully we will not be tempted to sacrifice one for the other and jeopardize consumer safety! Following the direction of the pharmaceutical industry and implementing a comprehensive risk assessment study is one way of minimizing these risks. It remains to be seen whether this approach will be taken up by individual manufacturers, driven by state regulations, or perhaps even addressed nationally as was done with USP 232 and ICH Q3D and driven through a regulatory/industry consortium established with the goal of providing minimum national standards for cannabis products in any of their forms. Federal scrutiny will clearly impact this decision, but it will be interesting to see how quickly this will happen. Only time will tell! ACKNOWLEDGEMENT I would like to recognize the contribution of Dr. Tony Destefano for helping me write the original white paper on the topic (10). He was president of the General Chapters and Healthcare Quality Standards at the United States Pharmacopeia (USP) who oversaw the development of general chapters 232 and 233 as well as being the USP observer to the ICH Q3D Expert Working Group. Tony’s knowledge on the inner workings of the USP was invaluable in developing this presentation. REFERENCES

1. Marijuana Policy by State: https://www.mpp.org/states

2. The Importance of Measuring Heavy Metal Contaminants in Cannabis and Hemp, R. Thomas White Paper, Analytical Cannabis, https://cdn.technologynetworks.com/ac/Resources/pdf/the-importance-of-measuring- heavy-metal-contaminants-in-cannabis-and-hemp-312957.pdf

3. A Recap of ASTM’s Workshop on Measuring Elemental Contaminants in Cannabis and Hemp Consumer Products, R. Thomas, Analytical Cannabis, August 2021 https://www.analyticalcannabis.com/articles/a-recap-of-astms-workshop-on-measuring- elemental-contaminants-in-cannabis-and-hemp-consumer-313229

4. NIST Tools for Cannabis Laboratory Quality Assurance, NIST CannaQAP Program,

https://www.nist.gov/programs-projects/nist-tools-cannabis-laboratory-quality-assurance

1. United States Pharmacopeia General Chapter <232> Elemental Impurities – Limits: First Supplement to USP 40–NF 35, 2017, https://www.usp.org/chemical- medicines/elemental-impurities-updates

2. United States Pharmacopeia General Chapter <233> Elemental Impurities – Procedures: Second Supplement to USP 38–NF 33, 2015, https://www.usp.org/chemical- medicines/elemental-impurities-updates

3. ICH Guideline Q3D on Elemental Impurities (R1), European Medicine Agency Website: https://www.ema.europa.eu/en/documents/scientific-guideline/international-conference- harmonisation-technical-requirements-registration-pharmaceuticals-human-use_en- 32.pdf

4. China Cashes in on the Cannabis Boom, New York Times editorial, S. Meyers, May 4, 2019, https:// www.nytimes.com/2019/05/04/world/asia/china-cannabis-cbd.html

5. China: Toxic Trails from Metal Production Harms Health of Poor Communities Amid Soaring Global Demand for Gadgets; G. Shih, Washington Post, January 5, 2020, https://www.business-humanrights.org/en/latest-news/china-toxic-trails-from-metal- production-harms-health-of-poor-communities-amid-soaring-global-demand-for-gadgets- 2/

6. Understanding Sources of Heavy Metals in Cannabis and Hemp: Benefits of a Risk Assessment Strategy, R. Thomas, A, Destefano, Analytical Cannabis, July 21, 2022, https://www.analyticalcannabis.com/white-papers/understanding-sources-of-heavy- metals-in-cannabis-and-hemp-benefits-of-a-risk-assessment-strategy-314044

Quality Standards in State Programs Permitting Cannabis for Medical Uses Authors: Cassandra L. Taylor, Schuyler A. Pruyn, Qiang Wang, Charles G. Wu; Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD ABSTRACT SUMMARY Currently in the United States, there exists a patchwork of state-level laws and regulations surrounding cannabis use. Although cannabis (excluding hemp under the Agricultural Improvement Act of 2018, Public Law 115-334) is illegal at the federal level and is not FDA- approved for any indication, many states allow patients with qualifying conditions to register for their medical cannabis program (MCP). To better understand the quality of cannabis found in these programs, we collected laws, regulations, and guidance documents available on public, state-run websites and compared them to Current Good Manufacturing Practices (CGMPs) applicable to finished drug products. CGMPs for human drugs contain minimum requirements for the methods, facilities, and controls used in manufacturing, processing, and packaging of a drug product to assure it is safe for use. Such a comparison will aid the development of consistent quality standards that could, in turn, improve the quality of a wide range of cannabis medical products in development that may be sold in the United States. States may likewise choose to have the cannabis and cannabis-derived products (CCDPs) that fall within their MCP follow quality-focused guidelines, such as those listed in CGMPs, to ensure the quality of these products and promote public health. This work further solidifies the need for standardized testing protocols and methodologies to keep consumers safe. INTRODUCTION There continues to be an increased public interest in studying cannabis for a variety of conditions such as seizure disorders, anxiety, and pain3. The U.S. Food and Drug Administration (FDA) regulates a wide variety of medical products, including drugs, foods, and dietary supplements. For drug development, several submission types4 exist in the Center for Drug Evaluation and Research (CDER)5 for researchers who want to study cannabis in clinical trials for various conditions. When CCDPs intended for use under human clinical trials are submitted, CDER treats these products like any other FDA-regulated product (i.e., subject to same authorities and requirements as FDA-regulated products containing any other drug substance)6-8. Separately, states have also been creating standards for use when regulating cannabis products under state authorities. While sharing a common goal of quality and consistency in products for consumers, there are differences between these standards and with the standards set out in the FDA guidance documents and CGMPs for drugs. This paper reports the results of our review of 37 available MCP regulations (see Supplementary Materials) from public, state-run websites. This article is not intended to be a comprehensive review of state quality requirements. The goal of this work was to (i) understand what quality-related tests are written into state regulations for CCDPs; (ii) compare and discuss the written testing regulations to those outlined in the drug CGMPs; and (iii) provide considerations to facilitate the development of consistent, high quality products for potential use in clinical trials of drugs. TEST METHOD/OVERVIEW On June 30, 2021, state-run websites (i.e., departments of health, cannabis-regulating offices, etc.) were visited to collect medical cannabis program laws, rules, regulations and/or guidance documents related to laboratory testing of cannabis and cannabis-derived products (see Supplementary Table 1). For each program, the most recent version of their laws, rules, regulations, and/or guidance documents found available on the website were downloaded and saved. Results were found for 37 state-level medical cannabis programs, which were included in the final analysis.

For each of the 37 programs, the identified tests included in their laws, rules, regulations, and/or guidance documents were summarized. From this summary, ten different tests were identified across 37 programs: heavy metal, pesticide, microbial, mycotoxin, residual solvent, foreign matter, water activity, moisture content, cannabinoid, and terpene (see Supplementary Materials, Definitions). Further analysis of the ten tests was completed by collecting lists of analytes to be tested for, as well as action levels for each. The information was then compared to Current Good Manufacturing Practices for human drug products. Microsoft Excel was used for all data recording and analysis. RESULTS/DISCUSSION To better understand the quality of CCDPs within state MCPs, we analyzed how closely the written regulations follow CGMPs. FDA’s portion of the Code of Federal Regulations is in Title 21, which interprets the Federal Food, Drug, and Cosmetic Act and related statutes. Parts 210 9 and 21110 contain CGMP regulations that must be followed by drug manufacturers. We looked specifically at 3 considerations within Part 211 Subpart I, which focuses on laboratory controls. These are identity and strength of active ingredients, appropriate laboratory testing for microorganisms, and acceptance criteria for quality control sampling and testing. As a general requirement, “laboratory controls shall include the establishment of scientifically sound and appropriate specifications, standards, sampling plans, and test procedures designed to assure that components, drug product containers, closures, in-process materials, labeling, and drug products conform to appropriate standards of identity, strength, quality, and purity.” We found some of the written state regulations would not be sufficient to support a submission to FDA for human clinical trials. While our understanding of these regulations was based on their plain language, we understand that the requirements and implementation may vary from state to state. CONCLUSIONS There is increased public interest in using CCDPs from state-regulated dispensaries in human clinical trials. In this study, we examined testing regulations within state-level MCPs and compared them with the federal CGMP requirements for drug products used in clinical trials. Our goal is to highlight the differences between the regulatory approaches related to product manufacturing and quality and promote consistency where possible. Our analysis illustrates the variances between MCPs and drug CGMPs. We, as scientific and medical professionals, note that our review was not intended to be a legal analysis, and primarily focused on only the testing sections of these regulations. Therefore, our analysis might not paint the complete picture of how regulations are implemented in each program. Importantly, although compliance with CGMPs may help ensure the quality and consistency of products, following CGMPs does not ensure that the products are safe or effective for their intended use. The best way to explore the therapeutic potential of these products is through FDA’s investigational new drug program. ACKNOWLEDGEMENTS We thank microbiologist Dr. Marla Stevens-Riley (Office of Pharmaceutical Manufacturing Assessment, Office of Pharmaceutical Quality, CDER/FDA) for providing expert comments and review. This research was supported in part by appointments to the Research Participation Program at the Center for Drug Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. REFERENCES 1 Cannabis, Coca, & Poppy: Nature's Addictive Plants. Cannabis: History. http://www.deamuseum.org/ccp/cannabis/history.html.

1. 2 MPP. Medical Marijuana Patient Numbers. https://www.mpp.org/issues/medical- marijuana/state-by-state-medical-marijuana-laws/medical-marijuana-patient-numbers/ (2021).

2. 3 American For Safe Access. State and Territory Qualifying Condition Chart. https://www.safeaccessnow.org/condition (2021).

3. 4 U.S. Food and Drug Administration. Electronic Regulatory Submission and Review. https://www.fda.gov/drugs/forms-submission-requirements/electronic-regulatory-submission- and-review (2019).

4. 5 U.S. Food and Drug Administration. Center for Drug Evaluation and Research | CDER. https://www.fda.gov/about-fda/fda-organization/center-drug-evaluation-and-research-cder (2020).

5. 6 Applications for FDA Approval to Market a New Drug, 21 C.F.R. §314.

6. 7 U.S. Food and Drug Administration. Drugs@FDA Glossary of Terms. https://www.fda.gov/drugs/drug-approvals-and-databases/drugsfda-glossary- terms#:~:text=A%20drug%20is%20defined%20as,any%20function%20of%20the%20body (2017).

7. 8 U.S. Food and Drug Administration. Cannabis Clinical Research: Drug Master Files (DMFs) & Quality Considerations. https://www.fda.gov/drugs/cannabis-clinical-research-drug-master- files-dmfs-quality-considerations-09162020-09162020 (2020).

8. 9 Current Good Manufacturing Practice in Manufacturing, Processing, Packing, or Holding of Drugs; General, 21 C.F.R. §210.

10 Current Good Manufacturing Practice for Finished Pharmaceuticals, 21 C.F.R. §211 (2021).

Cannabis: Was It Tested? Why You Need to Know Authors: Uma Dhanabalan, Darcey Trescone; Global Health & Hygiene Solutions, LLC, Uplifting Health & Wellness, Cambridge, MA ABSTRACT SUMMARY Cannabis is a plant used for medical, spiritual, and industrial uses for thousands of years throughout the world. The multifunctional benefits and socioeconomic impact of cannabis today is a global issue and a public health concern. Cannabis is in Schedule I as an illegal drug in the United States of America, meaning that there is no medical use and potential for abuse. Today 41 States and the District of Columbia allow for medical use and 23 States allow for adult use of cannabis products. Since Cannabis is Federally illegal, this preludes the US Food & Drug Administration from its regulations and oversight. Global analytical standards for quality testing of cannabis and worker safety are lacking. The rise in cannabis companies and consumption increases the need locally and globally. Establishing standards and polices with implementation and governance for the cannabis industry can minimize and mitigate deadly outcomes. The cannabis plant (including hemp) can absorb heavy metals, pesticides, and other toxins from the soil, air, water, and substrates which it is exposed to. Which makes it a Bioaccumulator. While this is a beneficial attribute for cleaning up polluted land, this can also lead to absorption of unwanted contaminants in the customers’ end products. Contamination can occur during processing and or post processing. The most common contaminants in cannabis include microbes, mold, heavy metals, solvents, and pesticides. Human consumption of contaminants, even low levels over time have substantial sequelae, especially in the immunocompromised and vulnerable population. This can lead to malignancy, cancer, developmental issues, reproductive, neurological, and endocrine disorders. We are at a critical juncture in human history where we need to address the global and public health challenges regarding this and the time for us to enact cannabis industry standards is today. INTRODUCTION Cannabis is a plant used for medical, spiritual, and industrial uses for thousands of years throughout the world. The multifunctional benefits and socioeconomic impact of cannabis today is a global issue and a public health concern. Cannabis is in Schedule I as an illegal drug in the United States of America, meaning that there is no medical use and potential for abuse. Today 41 States and the District of Columbia allow for medical use and 23 States allow for adult use of cannabis products. Since Cannabis is Federally illegal, this preludes the US Food & Drug Administration from its regulations and oversight. Standards for testing of cannabis and worker safety are lacking. The rise in cannabis companies and consumption increases the need locally and globally. Establishing standards and polices with implementation and governance for the cannabis industry can minimize and mitigate deadly outcomes. The extent of cannabis contamination is not fully understood. OVERVIEW For the first time in US history, older adults age 65+ are projected to outnumber children by 2034. This aging population suffers from a variety of chronic conditions that impact cardiovascular, renal, hepatic functions and other diseases including cancer. Consumption of cannabis amongst the older adult population, including the vulnerable population and those who are immunocompromised, is on the rise, which can pose additional risks. We are facing an opioid epidemic and mental health issues; cannabis has been used to treat

both and is a much safer alternative. There have been zero deaths due to cannabis overdose. Dr. Uma Says® “Cannabis is an entrance to a better quality of life, an exit drug from pharmaceuticals, narcotics, alcohol, and nicotine.” “Safety first, do no harm.” Cannabis can bioaccumulate and can be an agent for phytoremediation to rid areas of pollutants including heavy metals, demonstrating the extent to which cannabis can absorb contaminants from the soil, air water and other substrates it is exposed to. With this beneficial attribute for cleaning up polluted land, this can also lead to absorption of deadly substances and the risk of contaminating products from the plant. The most common contaminants in cannabis include microbes, heavy metals, and pesticides. Heavy metals from surrounding substrate soil and through cross contamination during processing or post processing. Whether the plant is grown inside or outside does not matter. Pesticides are used to prevent pests and pathogens from infecting plants. Human consumption of pesticides, even low levels over time have substantial sequelae, including malignancy, developmental issues, reproductive, neurological, and endocrine disorders. Heavy metals, solvents and pesticides, regardless of the route of exposure, can accumulate in human tissue leading to oxidative stress, enzyme inhibition, DNA damage, gene expression, and mimicry. This can result in membrane damage, protein dysfunction, DNA impairment, cell death, and mutagenicity. Microbial contamination generally occurs during the improper preparation and storage of products. It can vary in the process; for example, if during harvesting, the cannabis is wet, during drying and/or storage under humid conditions can often lead to fungal infections, including powdery mildew, budworm, or mite issues. These days there are multiple methods of cannabis delivery, which require various extraction processes to produce the necessary consumer product. Solvents utilized in extraction that are unsafe for human consumption should be removed from the product for sale; however, insignificant amounts of these solvents, ex butane, can still be found in consumer cannabis products due to the lack of testing standards. DISCUSSION The endocrine system is complex and impacts many organs within the human body, including the GI system, heart, kidney, pituitary gland, adrenal gland, hypothalamus, parathyroid gland, thyroid gland, pineal gland, pancreas, thymus, and testes/ovaries. Heavy metals, pesticides, solvents, and other foreign substances found in cannabis consumer products, even in tiny amounts, can wreak havoc on health. As a standard, laboratories should be testing for the following: potency, solvents, foreign material, heavy metals, bacteria, mycotoxins, pesticides, and water activity. Consumer education is key to ensuring the public is knowledgeable about the risks of consuming cannabis products that do not follow quality standards. People using cannabis are at risk both medical and adult use. Individuals that are immunocompromised and in the vulnerable population are at greater risk. Consumption for many is a daily thing, so continuous exposure, even in insignificant amounts, puts the public at risk for acute and chronic condition. CONCLUSIONS We are at a critical juncture in human history where we need to address the global and public health challenges and the need for standards for the Cannabis Industry. This will include standards for both hemp products for production and processing. Worker safety standards are

needed based on exposures and job analysis, the need for personal protective equipment, testing, and surveillance. The time for us to enact cannabis industry standards is now. REFERENCES

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KEYNOTE #2: Development of the Chemical Analysis Approaches to Support the Evolving Cannabis Sector Authors: Robert O’Brien; Supra Research, Kelowna, British Columbia, Canada ABSTRACT SUMMARY The Cannabis sector industry is rapidly changing as legalization opens up around the world and regions that are now operating legally, mature and evolve new classes of products. Analytical methods and approaches will need to be developed in order to not only manage the current sector needs but also ones that will come in the future. With Cannabis 1.0 products, such as dried buds, simple oils and vape products, the analytical focus was initially on measuring phytocannabinoids and targeted contaminants. As Cannabis 2.0 products begin to enter the marketplace, namely edibles, beverages, topicals, and concentrates, the analytical focus turned to methods to measure phytocannabinoids in these more complex matrices, as well as, stability testing and issues such as THC label claim inflation have come more of a focus. Another set of important analytics metrics that need to be developed are those to monitor the pharmacokinetics of Cannabis actives. Initially, these will be needed to justify claims such as increased absorption of nanoemulsions and related technologies, but will be critical in the evolution of Cannabis 3.0 products that will include more targeted and tailored products. These will be used as medicines, sleep aids but also to enhance experience such sensory enhancement (taste, sound and colors). These products will deliver a more controlled dose of select cannabinoids and terpenes, based on the understanding of the metabolism of the actives as determined by GCMS and LCMSMS techniques.

Progress in Development of Traps Suitable for Collection of Electronic Cigarette Aerosols for Analysis of Metals Authors: R. Steven Pappas, Naudia Gray; Centers for Disease Control and Prevention, Atlanta, GA ABSTRACT SUMMARY Various methods have been used in successful and unsuccessful attempts to collect aerosols from electronic cigarettes over the last ten years for the purpose of assessing the possibility for potentially harmful exposures. Development of an appropriate trapping method for analysis of metals in aerosols is one of the more challenging tasks for inorganic analytical chemists. Concentrations of the potentially harmful metal constituents of the aerosols generated by these devices are very low, so analysts must avoid environmental contamination of trapped aerosol with metals from the collection devices and ancillary materials used during sample preparation. The issue of contamination makes the use of materials commonly used to trap the samples for organic constituents inappropriate for accurate and reliable measurement of metal constituents. In the search for appropriate trapping materials and methods, several approaches have failed due to other problems such as resistance to air flow that is necessary to activate the devices and form an aerosol. This presentation will review selected approaches to trapping aerosols for analysis of metal constituents with appropriate materials and problems that have been solved or that remain to be solved before development of an ideal high throughput method for aerosol metals analysis. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. INTRODUCTION Various methods have been used in successful and unsuccessful attempts to collect aerosols from electronic cigarettes over the last ten years for the purpose of assessing the possibility for potentially harmful exposures. Development of an appropriate trapping method for analysis of metals in aerosols is one of the more challenging tasks for inorganic analytical chemists. TEST METHOD/OVERVIEW Concentrations of the potentially harmful metal constituents of the aerosols generated by these devices are very low, so analysts must avoid environmental contamination of trapped aerosol with metals from the collection devices and ancillary materials used during sample preparation. The issue of contamination makes the use of materials commonly used to trap the samples for organic constituents inappropriate for accurate and reliable measurement of metal constituents. In the search for appropriate trapping materials and methods, several approaches have failed due to other problems such as resistance to air flow that is necessary to activate the devices and form an aerosol. In order to provide accurate and valid results of inorganic elemental analyses, sample preparation procedures require high metals purity solvents and trapping materials and the avoidance of glass materials. These issues will be discussed in considering different approaches, including glass and quartz fiber filters, membrane filters, impingers, and condensation tubing traps. RESULTS/DISCUSSION No present trapping system is ideal. Some traps contaminate samples. Some traps provide low aerosol recovery. Some traps have too much air flow restriction. Traps that do not contaminate samples generally provide low sample throughput.

CONCLUSIONS More work is needed for development of aerosol traps that provide high aerosol recovery, low air flow restriction, and do not contaminate samples.

Evidence of Metal Particles in Cannabis Vape Liquids Authors: Zuzana Gajdosechova1, Joshua Marleau-Gillette2, Matthew J. Turnbull2, Jeremy E. Melanson2, Duane C. Petts3, Simon E. Jackson3, Ashley Cabecinha4, Hanan Abramovici4, Andrew Waye4 1NRC Metrology, Ottawa, ON, Canada; 2NRC, Ottawa, ON, Canada; 3NRCan, Ottawa, ON, Canada; 4Health Canada, Ottawa, ON, Canada ABSTRACT SUMMARY Recent surveys indicate that cannabis vaping is a popular mode of cannabis use. Cannabis vaping involves the vaporization of a cannabis vaping liquid or solid via a vaping accessory constructed of various metal or other parts. An increasing number of reports advocate for expansion of regulated metals in cannabis vape liquids beyond As, Cd, Hg, and Pb to reflect the possibility of consumers’ exposure to the large number of metal contaminants found in these products. However, the metal analysis in cannabis vape liquids is challenged by poor precision and reproducibility. Herein we present results of 12 metal concentrations in 20 legally purchased and 21 illegal cannabis vape liquids. Cobalt, chromium, copper, nickel, lead and vanadium in several legal and illegal products exceeded the established tolerable limits, in some instances by > 100-fold. Lead mass fraction in 5 illegal samples reached μg g-1 and high levels of nickel (max 677 μg g-1), zinc (max 426 μg g-1) and copper (max 485 μg g-1) were also measured. Significant differences in metal concentrations were observed in cannabis vape liquids originating from two identical devices, although the liquid was of the identical lot. Metal particles were observed by scanning electron microscopy, and laser ablation inductively coupled plasma mass spectrometry confirmed presence of copper, zinc, lead and manganese particles. Co-localized particles containing aluminum, silica, and sodium were also detected. These results identify different metal particles as a possible cause of poor measurement precision and for the first time, provide evidence of metal particles in cannabis vape liquids contained in unused cannabis vape pens. INTRODUCTION Legalization and regulation of cannabis in Canada and at the state level across the US has led to a wide variety of commercially available cannabis products. Inhalation is still the dominant mode of cannabis consumption; however, the traditional smoking of dried cannabis through combustion has seen a decline while vaping of cannabis has seen an increase, particularly in young adults who are concerned with the health implications associated with smoking.1,2 Vaping cannabis liquids is a non-combustion process, where a cannabis liquid concentrate is aerosolized upon contact with a resistance-heated element and inhaled through a mouthpiece. The heating element is a metal wire which vaporizes the vape liquids directly, or through a cotton wick soaked in the vape liquid. Newer generation of coils, so called ceramic coils, incorporated ceramic as a wicking material and depending on the design, either completely replaced the cotton wick or are used in conjunction with it. There are a large variety of designs on the market that are based on the same vaporization principle; however, the individual components can be made of different metal alloys. The most commonly used are stainless steel (Cr, Ni, Fe, Mn), nichrome (Ni, Cr), Ni-plated brass (Cu, Zn), kanthal (Al, Cr, Fe) with tin (Sn) and lead (Pb) being used as solders.3,4 Several publications have shown that the metallic components of vaping devices leach metals into the vaping liquids5,6 and thus more research is needed to understand the risk associated with vaping. In this study, the metal content in cannabis vape liquids from 20 legal and 21 illegal electronic vaping devices were analysed and possible implications are discussed. Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDS) and laser ablation

inductively coupled plasma mass spectrometry (LA-ICP-MS) were used for in situ approaches to identify metal particles within cannabis vape liquids. The data presented are from unused vaping devices, hence, represent the baseline levels that the consumer may be exposed to. TEST METHOD/OVERVIEW The cannabis vape liquids were collected from their respective sealed cartridges and prior to subsampling, were warmed up in a hot block at 60 °C for 30 min and stirred with a pipette tip. An aliquots of approximately 0.1 g were accurately weighed into microwave digestion vessels and digested in 6 mL of concentrated HNO3 in a microwave digestion system. The digested samples were analyzed for total metals content using ICP-MS/MS. To investigate the presence of metal particles, a portion of 15 μL of the cannabis vape liquid was placed into the Quantomix QX-102 capsule and examined by SEM equipped with energy dispersive X-ray spectrometer. Element mapping of the cannabis vape liquid was carried out by -ICP-MS. RESULTS/DISCUSSION The concentrations of As, Hg, and Cd in all tested samples were within the generally accepted Schedule B publication tolerance limits of 0.2, 0.1, and 0.3 μg g-1, respectively but the concentration of Pb exceeded the tolerance limit of 0.5 μg g-1 in 1 legal (0.628 μg g-1) and 6 illegal samples (range 1.52 – 48.9 μg g-1). The mass fractions of measured metals in legal samples varied significantly and several samples with mass fraction of Ni above the established tolerable limit also yielded Cr and/ or Cu levels above established limits. Similarly, illegal samples high in Pb also measured high for Cu, Ni, and Zn. When compared with permitted levels of elemental impurities in inhaled products established by European Pharmacopoeia, several legal and illegal samples significantly exceeded established tolerance limits. While only few samples exceeded the limits for Co and V, large number of samples were above the limits for Cr, Cu, Ni and Pb. Very often, samples exceeded these limits in more than one measured analyte. The magnitude by which some of the samples exceeded these limits varied between the analytes, with Ni being 900 times above the established limits in several illegal samples. Additionally, due to limited amounts of some legal cannabis vape liquids (˂0.2 mL) the replicate analysis was done on liquids obtained from two devices purchased at the same time and from the same production lot. Surprisingly, their metal composition varied significantly as did their visual appearance, despite coming from the same production lot. The SEM analysis showed presence of metal particles within cannabis vape liquid. The elemental composition of these particles was significantly different from the composition of the QX-102 capsule suggesting that the metal particles originated from metal components in contact with the vape liquid while in the device and not the capsule. To corroborate the observations by SEM/EDS, a drop of cannabis vape liquid placed on a glass slide was analyzed by LA-ICP-MS. Hotspots were observed in the Cu, Pb, Zn, and Mn maps for this sample, with some co- localization between particles, indicating presence of particles containing these elements. The largest number of particles were observed in the maps for Pb followed by Cu. A small number of Zn and Mn clusters were observed but their signal intensity is similar to, or slightly higher than the matrix, suggesting that they are at the limit of particle size detection. CONCLUSIONS While it is true that compared to smoking, vaping reduces the exposure of the user to several toxicants and carcinogens such as polycyclic aromatic hydrocarbons, volatiles, and nitrosamines, there is ample evidence of exposure to metals in vape aerosols. The presented data from legally purchased and illegal cannabis vape devices showed mass fractions of Pb above the currently established tolerance limits in several of the vape liquids analyzed, particularly in the illegal samples where Pb concentrations were up to 100 times higher than the limit. Additionally, the measured mass fractions of toxic metals such as Cr, Cu, Ni, and Co, as well as the essential metals Zn and Mn that have known inhalation toxicity, add to the existing evidence that long

term vaping may carry risks to health. More importantly, the use of imaging techniques SEM/EDS and LA-ICP-MS confirmed the presence of metal particles in studied samples. Previous studies suggested that metal particles may be released from the metal coils during the heating cycles to generate aerosols; however, our data showed that metal particles are present in the cannabis vape liquids at the point of purchase, before their actual use. The origin of these particles is unknown. Further research studies of vape devices are necessary to better understand the composition of the metal parts of the devices as well as other factors that promote leaching of metals into the liquids (e.g., storage temperature, pH). Given the analytical challenges encountered in the present study, further method development is needed for particle detection by SEM-EDS and LA-ICP-MS. While these techniques provided pertinent information about the identification and distribution of metals particles, their full potential could not be utilized due to the physical properties of cannabis vape liquids. Making more information about the metal components of vape device available along with the filling date of the vape device can help support and inform additional research studies and risk assessments. Development of standards for vaping device construction and the materials used could also be considered by standards development organizations to reduce the risks of metals leaching into the vaping liquids. ACKNOWLEDGEMENTS MJT would like to acknowledge Jeff Fraser, Oltion Kodra, and David Kingston, for their valuable discussions and inputs in the SEM analysis. Ontario Provincial Police is acknowledged for providing illegal cannabis vape liquid samples. REFERENCES (1) Shiplo, S.; Asbridge, M.; Leatherdale, S. T.; Hammond, D., Medical cannabis use in Canada: vaporization and modes of delivery. Harm Reduct J 2016, 13 (1), 1-10. (2) Chadi, N.; Minato, C.; Stanwick, R., Cannabis vaping: Understanding the health risks of a rapidly emerging trend. Paediatr Child Health 2020, 25 (Supplement_1), S16-S20. (3) Lynch, J.; Lorenz, L.; Brueggemeyer, J. L.; Lanzarotta, A.; Falconer, T. M.; Wilson, R. A., Simultaneous Temperature Measurements and Aerosol Collection During Vaping for the Analysis of Δ9-Tetrahydrocannabinol and Vitamin E Acetate Mixtures in Ceramic Coil Style Cartridges. Front Chem 2021, 9. (4) Gray, N.; Halstead, M.; Gonzalez-Jimenez, N.; Valentin-Blasini, L.; Watson, C.; Pappas, R. S., Analysis of toxic metals in liquid from electronic cigarettes. Int J Environ Res Public Health 2019, 16 (22), 4450. (5) Olmedo, P.; Goessler, W.; Tanda, S.; Grau-Perez, M.; Jarmul, S.; Aherrera, A.; Chen, R.; Hilpert, M.; Cohen, J. E.; Navas-Acien, A., Metal concentrations in e-cigarette liquid and aerosol samples: the contribution of metallic coils. Environ Health Perspect 2018, 126 (2), 027010. (6) Pappas, R. S.; Gray, N.; Halstead, M.; Valentin-Blasini, L.; Watson, C., Toxic metal- containing particles in aerosols from pod-type electronic cigarettes. J Anal Toxicol 2021, 45 (4), 337-347.

Latex is a Terpene: Why Aren’t We Looking for Macromolecules in Cannabis Vape Oils and Vape Condensates? Authors: Daniel A. Batzel; Polyene Services of California, Inc, Walnut Creek, CA ABSTRACT SUMMARY Cannabis vape oils are incompletely characterized. About 7% by weight is unknown because state cannabis testing rules don’t require total characterization like the FDA would require a company to demonstrate for a new food or drug substance. Vape oils comprise olefins (molecules with double bonds). In fact, THC and CBD are olefins. Commercial products comprising olefins are often sold stabilized or refrigerated. Turpentine is stabilized (usually with vitamin E acetate), otherwise it will thicken and eventually form a brick in the can due to addition reactions between the terpenes. Edible oils comprising unsaturated fats are usually stabilized to prevent addition reactions (otherwise they thicken due to crosslinking addition reactions and their pour points undesirably increase). Butter does not have stabilizers; instead, it is sold refrigerated to prevent hardening due to addition reactions. Old fashioned varnishes (before synthetic alkyd resins) were mixtures of terpenes and unsaturated fats. They didn’t comprise stabilizers of course because the purpose of a varnish is to crosslink and form a hard coating. Are molecules comprising cannabis vape oils undergoing addition reactions on dispensary shelves and forming macromolecules? When vape oils are heated, do any of the olefins therein undergo addition reactions? Is it a bad assumption to think that molecules “break down” (in size) during vaping, when they might be getting bigger? What harms might there be to inhaling molecules with a propensity to undergo addition reaction?

Understanding Thermal Degradation Products as Contaminants Generated by the Chosen Introduction Approach Authors: Robert O’Brien, Seamus Riordan-Short, Ryan Hayward; Supra Research, Kelowna, British Columbia, Canada ABSTRACT SUMMARY The fundamental reason for testing consumer products for contaminants is to ensure, when the products are used as intended, that consumers will not be exposed to chemical agents that can cause them harm. In the case of Cannabis products, a majority of products are sold with the intention of being consumed by inhalation of combustion products or by inhalation of vapors generated after heating materials to elevated temperatures. In both of these cases, the chemical profile of the chemical agents that will enter the consumer's body can be fundamentally different from what is present in the purchased product. If consumers are to be protected from exposure to dangerous chemical agents, then thermal degradation and related reactions need to be understood and characterized. Unlike tobacco, cannabis can be prescribed as a medicinal product, and in that context, it is critical to understand the dose of medicinally active ingredients that are delivered to the patient. This presentation will focus on GCMS techniques used to measure what chemical agents can be generated at different temperatures as well as techniques to characterize vapors from a variety of cannabis products. INTRODUCTION The consumption of Cannabis materials by inhalation involves heating the material in some manner to make the active Phytocannabinoid compounds and terpenes volatile enough to be inhaled and absorbed into the bloodstream of the consumer typically through the capillaries in the lungs. Historically heating was most often accomplished by combustion of the plant material often enclosed in paper, in other words smoking a cigarette or a joint. Combustion generates a complex plume of chemical agents that include not only the phytocannabinoids, terpenes, and other semi-volatile components in the cannabis but also thermal and combustion byproducts of the biomass. It is generally believed that heating cannabis material in a vaporization device set at temperature well below the threshold required for combustion generates less harmful degradation byproducts and related chemical agents. However, the profile of the chemical agents produced by the heating process is both temperature dependent and dependent on the other chemical compounds that can potentially lead to a reactive mixture. Specifically, the presence of additives, flavors and material can impact the types of chemical agents produced. Early in 2019 a series of hospitalizations and deaths were associated with e-cigarette or vaping use-associated lung injury (EVALI). The exact cause of these events is unknown but there was a strong association with Vitamin E acetate used as a thickening agent. There was also evidence that other compounds that could be safely ingested because they were classified as Generally Regarded as Safe (GRAS) could produce dangerous chemical agents when heated to elevated temperatures. From this it became evident that some ingredients or formulations could generate dangerous chemical agents when heated in vaporization devices, but there currently is no accepted protocol to screen such contaminants and the development of appropriate exposure safety limits can take considerable time to secure scientifically and regulatory consensus. This research describes a testing-based approach to screen for dangerous ingredients and formulations using a well-defined heating process in a headspace vial followed by an analysis of the residual solvents generated by this heating process. There are well established limits for the concentrations of common residual solvents in commercial products and if the concentration of these compounds exceeded the permitted threshold for residual solvents, the ingredient or

formulation would be deemed inappropriate for use at the set incubation temperature where these chemical agents are generated. TEST METHOD/OVERVIEW Ingredients or formulations being evaluated for use in Heated Vaporization products were sealed in 10 mL headspace vials and heated to a variety of temperatures and held for 5 minutes to enable thermal degradation and oxidation generated chemical agents to form. These vials were then cooled to room temperature and analyzed using a modified residual solvent method USP <467>. The presence of any observed residual solvents above the acceptable limits defined in USP <467> was then used to determine if the ingredient or formulation was suitable for use at the evaluation temperature. RESULTS/DISCUSSION Using the described approach, we were able to successfully screen a variety of potential additives proposed for use in Heated inhalation products. We selected 240C as a maximum temperature for vaping and using that temperature we were able to show that many of the suspected substances degraded to such high levels of residual solvents that they would fail a residual solvent analysis after being heated to 240C for 5 minutes. Vitamin E acetate, Phytol and Squalene all degraded to very similar residual solvents including formic acid, acetic acid, acetone, and a variety of other oxidation products. The analysis of Phytol indicated that it would be problematic 6 months earlier than it was shown to cause deaths in rats. CONCLUSIONS This approach provides a convenient and cost-effective screening method as a stress test for specific ingredients and formulations that degrade to dangerous chemical agents on heating eve though these compounds are regarded as GRAS.

From Back to the Land to Burying the Land Authors: Mitchell Colbert, Crockett, CA ABSTRACT SUMMARY In the early days, the cannabis legalization movement was heavily linked to the environmental movement, to the point where early activists “never expected waste to be an issue.” One big game changer was when ecigarette technology was appropriated to create cannabis “vape pens,” more accurately referred to as an Electronic Cannabis Delivery Systems (ECDS). With the creation of ECDS, a waste stream that was largely compostable, shifted to become a waste stream where ewaste (ECDS and batteries) represented 14-32% of total sales depending on the year and state. The other major issue was regulations around cannabis waste that frequently were so poorly written they overlooked cannabis ewaste, resulting in contamination of landfill waste, soils, and water. Regulators imposed requirements like rendering waste “unusable and unrecognizable,” which prevented any chance of recycling and may violate hazardous waste disposal rules due to heavy metal contamination of landfill waste. Despite rules around cannabis waste recycling being ambiguous at best, or draconian at worst, many businesses do engage in the collection of vape waste for recycling and other sustainable practices. Many of the waste issues seen in the cannabis industry today are also present in the hemp industry. The hemp industry does face an additional barrier, which is the unreasonably low 0.3% THC threshold, resulting in over 20% of the US domestic hemp crop being destroyed or remediated every year not because of disease or drought, but federal regulations because it tested “hot.” Disposal options for that hot hemp are limited and do not offer farmers all the remediation options needed for their crop, such as using it to make bioplastics. Regulations must be changed at the state and federal levels to allow for better cannabis and hemp waste management options resulting in less environmental contamination. INTRODUCTION The cannabis industry is a rapidly growing industry that produces a very large volume of waste compared to other FMCGs, including ewaste in the form of ECDS. While smoking cannabis was the predominant method of consumption with over 50% of sales being cannabis flower in most legal markets, in recent years there has been a strong shift away from smoking cannabis towards vaping it or consuming it in other methods that require more processing and packaging. These other methods of consumption have created new and diverse waste streams, which have been made more complicated by regulations requiring childproof packaging, universal warning symbols, warning labels, and all manner of other strict requirements that have led to larger cannabis packages than before legalization, and thus more waste since legalization due to the regulations on packaging. Much of this packaging waste and ewaste is then mandated to be disposed of at landfills due to sloppily written regulations, that may violate other waste rules at the state and federal level. TEST METHOD/OVERVIEW Testing methodologies will combine a literature review of available information about the cannabis and hemp markets and waste disposal options available with surveying of cannabis operators to determine the rate of on-site waste recycling and other sustainable practices. Additionally, a data set of over 200 cannabis products and over two dozen hemp products was generated and analyzed to the determine the average product waste weights to estimate the volume of the total waste stream in California.

RESULTS/DISCUSSION The preliminary 2019 survey of legal dispensaries in California found that 53.8% had takeback containers on site to collect vape batteries and cartridges for the purpose of recycling or responsible disposal. While half of dispensaries were collecting waste on site for recycling, this was being done in a regulatory environment where many businesses’ read of the regulations is one that views recycling on site as being non-compliant and a risk if they were audited. This regulatory ambiguity has led directly to pollution of soils and groundwater in California as ewaste has been ground up and sent to the landfill, simply because it contains some cannabis residue. An updated online survey conducted in 2023 with all cannabis operators, not just dispensaries, found that the overall rate of onsite recycling was 85.7%, but the rate of cannabis waste recycling was just 64.3% Research on other FMCG industries has shown that the average packaging weight is 9% of the product weight, compared to cannabis, where the only market category where the average packaging weighs less than the product itself is edibles. As every cannabis market segment other than edibles, produces more waste than products, they are effectively waste businesses that happen to also sell products, rather than companies who sell products that happen to produce waste. Regulations must be changed to allow cannabis companies to package their products like all other FMCG industries and to recycle their waste like other FMCG industries, otherwise, cannabis sustainability will remain a myth. Additionally, regulators should create a cannabis waste hauler license (like the one that exists in Mississippi) to ensure waste is more effectively and safely processed. At the federal level, IRS Tax Code 280E must be eliminated, as it hamstrings any potential profits that could be spent on making the business more sustainable. On the hemp side, 20% of the yearly crop testing hot and being destroyed or remediated is an unacceptable waste of millions of gallons of water and countless other resources. Activist groups estimate that it could be as high as 50-60% of some state crops being unusable for various reasons. Rather than destroying it, that hemp should be fully remediated into biofuels, bioplastics, livestock feed, or other uses. If we want this industry to return to being back to the land, and drop their current practices of burying the land in packaging waste, that change can only come from the people who created the regulations and statute that have tied the cannabis industry’s hands. CONCLUSIONS While the cannabis and hemp industries were intended to be sustainable, environmentally friendly industries, due to regulations and statute governing their commerce, businesses have their hands tied in a statutory straitjacket. While companies want to be more sustainable, in many cases using less packaging is not a legally compliant option. Cannabis companies also face additional tax burdens unheard of for all other FMCG industries, which limits their available funds to switch to more environmentally friendly sustainable packaging even when allowed by regulations. Hemp farmers need better options for hemp waste disposal, such as using it to make bioplastics or animal feed, but those changes will need to come from the USDA, which may be a heavier lift than state governments cleaning up their cannabis waste streams, but it would have a much broader impact. ACKNOWLEDGEMENTS I would like to thank all of the cannabis and hemp businesses who took part in my surveying of on-site recycling and my online survey, all the cannabis companies and consumers who helped create my dataset of packaging weights, and all the trailblazing activists who had hoped to create a more sustainable cannabis industry than the one we presently have. I would like to acknowledge the hard work and sacrifice of all the cannabis and hemp farmers who struggle to make ends meet more than your average farmer and who generally lack access to banking, loans,

and crop insurance. If it were not for the world’s farmers, we would be starving, naked, and suffering from a lack of biopharmaceuticals. We owe our modern society to our farmers more than any coder or tech entrepreneur. I would additionally like to thank all of my professors who expressed apprehension at my insistence on focusing on cannabis research, especially the one who cautioned me against becoming “the cannabis guy,” it is thanks to your wonderful teachings that I am where I am today. REFERENCES California Department of Tax and Fee Administration. Cannabis Tax Revenues. https://www.cdtfa.ca.gov/dataportal/dataset.htm?url=CannabisTaxRevenues (September 25th, 2023). Cowee, Maggie. 2019. “Chart: 20% of hemp lots will exceed 0.3% THC limit next year, USDA estimates.” Hemp Industry Daily, December 12th. https://hempindustrydaily.com/chart-20-of- hemp-lots-will-exceed-0-3-thc-limit-next-year-usda-estimates/ (September 25th, 2023). (September 25th, 2023). (September 25th, 2023). Fery, Blake. 2022. “Exploring category shifts in the California cannabis market.” Headset. https://www.headset.io/blog/exploring-category-shifts-in-the-california-cannabis-market (September 25th, 2023). NCV Newswire. 2018. “California Cannabis Market Trends Emerge.” New Cannabis Ventures, August 30th. https://www.newcannabisventures.com/california-cannabis-market-trends-emerge/ (September 25th, 2023). United Nations. Economic and Social Commission for Western Asia. https://archive.unescwa.org/fast-moving-consumer-goods (September 25th, 2023). David J. Smith, Eric M. Serum, Thomas M. Winders, Bryan Neville, Grant R. Herges, Carl R. Dahlen & Kendall C. Swanson (2023). Excretion and residue depletion of cannabinoids in beef cattle fed hempseed cake for 111 days, Food Additives & Contaminants: Part A, 40:4, 552-565, DOI: 10.1080/19440049.2023.2187645 Eva Pongrácz (2007). The Environmental Impacts of Packaging. In Environmentally Conscious Materials and Chemicals Processing, ed. Myer Kutz. Wiley. https://doi.org/10.1002/9780470168219.ch9

Preventing Contamination: The Role of Good Manufacturing Practices (GMPs) Authors: Steven Gendel; Gendel Food Safety LLC, Silver Spring, MD ABSTRACT SUMMARY Cost-effective contamination control for cannabis/hemp-derived products depends on preventing problems rather than relying on expensive and disruptive responses after contamination has happened. The essential foundation of prevention is a suitable, safe, and well-controlled production environment. Good Manufacturing Practices (GMPs) are the practices and controls that provide this environment. Further, GMPs are required prerequisites for the HACCP and Preventive Controls systems that are mandated in some state regulations. Regulatory agencies and industry groups have defined specific GMP requirements for traditional products (including foods, dietary supplements, and medicines) that are all based on a common set of principles that can be applied in the cannabis/hemp industry. These principles include controls related to the facility, operations (including cleaning and sanitation), personnel practices, equipment, suppliers, environmental monitoring, and documentation. It is essential that the cannabis/hemp industry define and implement industry specific GMPs to ensure quality and sustainability and to support the development of nationally harmonized regulatory systems. INTRODUCTION The presence of contaminants in cannabis products can harm consumers and businesses and may lead to regulatory actions against the company that made the product. Preventing contamination avoids these problems and is much more effective than responding after contamination has happened, Good Manufacturing Practices (GMPs) describe the conditions and actions that are needed to produce contamination-free products. TEST METHOD/OVERVIEW Not applicable. RESULTS/DISCUSSION ASTM Committee D37 is preparing a Standard Guide for GMPs for cannabis edibles, dietary supplements, topicals, and inhaled products. When finalized, this standard will address GMP practices for buildings and grounds, personnel, operations, equipment, processes, suppliers, recalls, and documentation. CONCLUSIONS The ASTM Standard Guide has benefitted from input from many members of Committee D37. The final version of the guide will undergo balloting soon and should be ready for publication early next year. ACKNOWLEDGEMENTS Thanks to David Vaillancourt, Kathleen May, Kathy Knutson, Steve Cooper, and James Farrell. REFERENCES Not applicable.

USP Perspectives on the Limits of Contaminants in Cannabis Authors: Nandakumara Sarma; United States Pharmacopeial Convention, Rockville, MD ABSTRACT SUMMARY Interest in cannabis-derived products is active and growing. Reports indicate that poor quality products can lead to acute adverse events and long-term health risks as well as lack of the expected benefits. Also, ensuring quality is important for conducting clinical research related to the development of drugs containing cannabis and cannabis-derived compounds. Appropriate quality specifications, including analytical methods and acceptance criteria, help ensure identity, composition, and minimal exposure to contaminants. Controlling contaminants is critical for cannabis, since it is known to readily absorb inorganic elements, including heavy metals, and is also subject to contaminants such as pesticide residues, microbial load, Aspergillus and aflatoxins. The United States Pharmacopeia (USP) Cannabis Expert Panel published cannabis-specific information on quality specifications, which can be a helpful resource for information on quality considerations for cannabis.1 USP had also published a proposal for monograph for cannabis inflorescence in the Herbal Medicines Compendium and a proposal for an informational General Chapter <1568> on cannabis quality for clinical research. The recommended limits for contaminants include risk-based acceptance criteria and analytical methodologies based on the USP general chapters. These standards could be used as a resource for toxicologically based limits for contaminants to control quality. 1 See Sarma ND, Waye A, ElSohly MA, Brown PN, Elzinga S, Johnson HE, Marles RJ, Melanson JE, Russo E, Deyton L, Hudalla C, Vrdoljak GA, Wurzer JH, Khan IA, Kim N-C, Giancaspro GI. Cannabis Inflorescence for Medical Purposes: USP Considerations for Quality Attributes. J Natural Products 83 (4), 1334-1351, 2020, at https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b01200.

Fungal Contamination Monitoring in Legal Cannabis Products: Optimization of Detection and Quantification Method for Mycotoxins in Cannabis Inflorescence and Edible Oil Using UHPLC-MS/MS Authors: Vincent Desaulniers Brousseau1,2, Emannuelle Bahl1, Julie Lacroix-Labonté2, André Robichaud3, Mark G. Lefsrud3 1Health Canada, Montréal, QC, Canada; 2McGill University, Montréal, QC, Canada; 3McGill University, Sainte-Anne-de-Bellevue, QC, Canada ABSTRACT SUMMARY Health Canada’s mission to maintain and improve Canadian’s health requires rigorous testing of cannabis legal products. Cannabis products contaminants need to meet the requirements set out in the Cannabis Act and Regulations. Mycotoxins are known cannabis contaminant. They are secondary metabolites produced by fungi that, when exposed to humans, can cause serious health issues. Numerous fungal species have been detected on cannabis. Notably, the toxigenic Fusarium spp, Aspergillus spp and Penicillium spp. These fungal species produce aflatoxins, ochratoxin A and deoxynivalenol. In order to ensure product safety for Canadian consumers, a detection and quantification HPLC-MS/MS method was devised for these mycotoxins. Contrary to other published methods, this one does not require costly immunoaffinity columns or isotope- labeled internal standards. The method was validated on multiple samples of cannabis plants and edible oil. It was shown that accurate quantification in plant samples needed a sample-matched calibration curve. A solvent calibration curve was sufficient for accurate quantification in edible oil samples. Limits of detection and quantification were sufficient for regulatory and monitoring purposes. This method could decrease compliance-related costs for the legal cannabis industry. INTRODUCTION Mycotoxins are metabolites produced by certain fungi as they grow in plant biomass. Poor agricultural practices and storage conditions cause the accumulation of these toxins1. The regulations on aflatoxin levels in cannabis set out in the Cannabis Regulations2 are based on the standards for natural health products by the Natural Health Products and Non-Prescription Products Directorate (NHPD)3. Although only aflatoxins are regulated, Health Canada's Cannabis Laboratory is developing tests capable of identifying and quantifying ochratoxin A and deoxynivalenol for monitoring purposes, in keeping with its mission to protect the health of Canadians. Similarly, the United States Pharmacopeia has identified AFs, OTA and DON as mycotoxins of concern in cannabis1. This is because when ingested, they can cause health problems, collectively referred to as mycotoxicoses. Mycotoxicoses can cause serious problems to the digestive, reproductive, nervous, circulatory and other systems4,5. Specifically, aflatoxins can cause liver cancer (hepatocarcinoma) and suppress the immune system. OTA is nephrotoxic and suspected carcinogen6. DON causes gastric distress and vomiting5. TEST METHOD/OVERVIEW An overview of the specific modifications made to the LC-MS-MS method is summarized. Extraction solvent composition, solvent to cannabis product mass ratio, injection volume and mass spectrometry parameters have been fine-tuned for detections of mycotoxins. Six mycotoxins were targeted: aflatoxins (G1, G2, B1 and B2), ochratoxin A (OTA) and deoxynivalenol (DON).

RESULTS/DISCUSSION Aflatoxins can be quantified in the range of interest (<20 ppb). OTA and DON can now be quantified in the same range that are applied to food and natural health products: <200 ppb and <1000 ppb respectively. Analyst exposure time has been reduced by one hour for a 10-sample run, while solvent ruse has been reduced by 50%. The need for matrix-match standard curve was proven necessary as seen it greatly increase accuracy of the method as seen in table 1. Table 1. Average values of recovery rate when quantification is made with a matrix-match curve (MM) versus a standard curve in a mix of methanol and water (diluant) MM (N=25)

1. AFB1 85.3±13.7

2. AFB2 82.5±7.6

3. AFG1 90.1±9.2

4. AFG2 93.7±16.8

OTA 75.7±15.4 DON 101.0±8.5 CONCLUSIONS Diluant (N=25) 67.2±12.0 71.0±7.7 77.1±9.3 79.2±14.0 70.4±23.2 73.8±9.1 The simplified method shows an interesting potential for the detection of regulated mycotoxins (AF) and for those monitored (OTA and DON). The recovery rates are satisfactory. The amount of solvent used was reduced by half and the exposure to the analysts was reduced by about 1h for a batch of 10 samples. The expansion of the method to other matrices of cannabis product types and other mycotoxins of increasing concerns is one example of how HC CL contributes to provide efficient testing for regulatory and surveillance purposes. REFERENCES

1. Sarma, N. D. et al. Cannabis Inflorescence for Medical Purposes: USP Considerations for Quality Attributes. Journal of Natural Products vol. 83 1334–1351 (2020).

2. Goverment of Canada. Cannabis Act (S.C. 2018 c.16). Justice Laws Website https://laws- lois.justice.gc.ca/eng/acts/c-24.5/ (2018).

3. Health Canada. Quality of Natural Health Products Guide Natural and Non-prescription Health Products Directorate. https://www.canada.ca/content/dam/hc-sc/migration/hc- sc/dhp-mps/alt_formats/pdf/prodnatur/legislation/docs/eq-paq-fra.pdf (2015).

4. Charmley, L. L. & Trenholm, H. L. Regulatory Guidance: Contaminants in Feed - Animal health. Canadian Food Inspection Agency Fact Sheet https://inspection.canada.ca/animal- health/livestock-feeds/regulatory-guidance/rg- 8/eng/1347383943203/1347384015909?chap=1 (2017).

5. Richard, J. L. Some major mycotoxins and their mycotoxicoses-An overview. Int. J. Food Microbiol. 119, 3–10 (2007).

6. Mondoly, P. & Poncelet, J.-L. Les Mycotoxicoses. Société Natl. des Group. Tech. vétérinaires (2005).

Market Audits Combat Cannabis Misinformation Authors: Jeff Rawson1, Lacey Keller2, Tori Turkington1, Jamie Toth3 1Institute of Cannabis Science, Cambridge, MA; 2MK Analytics, Denver, CO; 3Eye on Cannabis, Oregon ABSTRACT SUMMARY We demonstrate that cannabis products may often be mislabeled, and we show how active monitoring through off-the-shelf testing and forensic data analysis can reduce this misinformation. We tested products off the shelves of dispensaries in MA for their content of cannabinoids and microbial contaminants. Our results showed that levels of cannabinoid compounds are systematically over labeled, and that microbial contaminants reach consumers. We demonstrate how the analysis of aggregated test results for discontinuities in the frequencies of values can pinpoint manipulation of data. Our results demonstrate a strategy for actively monitoring a market for cannabis to make consumers more safe and better informed. INTRODUCTION Consumers of cannabis trust third-party testing to ensure the safety and contents of products, but false test results have been discovered in several documented cases.1-4 One independently- funded study of cannabis flower from California dispensaries found 87% contained levels of psychotropic 9-tetrahydrocannabinol (THC) lower than labeled by >10%.3 Cannabis flower is tested for pesticides, heavy metals, microbiological contaminants, and content of cannabinoid compounds, but the reliability of these Quality Control (QC) tests is unproven. Medical consumers and doctors require knowledge of contents for dosing cannabis and evaluating risks,5 and researchers need accurate data to measure the impacts of legalization. Consumer confidence in the cannabis markets of many states is low. The over labeling of content of THC has earned the moniker ‘THC inflation,’ or ‘potency inflation’ since the levels of many cannabinoid compounds matter to consumers. We demonstrate forensic data analysis of test results for monitoring cannabis markets. The results of every lab test of cannabis performed in most states are recorded in a database called a “seed-to-sale” system. Labs perform measurements that are subject to natural variance, and their natural origin suggests that they should fall on curves that are relatively continuous; but if humans manipulate the results of many tests, we may detect artifacts such as discontinuities.2 We demonstrate how assays for discontinuities in the frequencies of test results may detect fraud. We also interrogate datasets for lab shopping, such as a cultivator who consistently tests with one lab, then changes to a new lab with a simultaneous improvement in the average value of potency tests or the rate of QC failures. TEST METHOD/OVERVIEW Testing off-the-shelf: We purchased 15 products from seven dispensaries in the Greater Boston area: three from one and two from the rest. We selected flower labeled 30+ %THC. When products above that threshold were not available, we bought flower recommended by dispensary staff. We divided each product into thirds that we delivered to cannabis testing labs. Three labs determined concentrations of cannabinoid compounds (Total Active Cannabinoids, TAC [wt%]) for 15 products, and one lab culture plated 10 of the products for microbiological contamination. The labs were aware of our study but blind to all details. We analyzed the results with R Studio. The TAC results for each sample were compared for consistency between labs via one-way ANOVA, and averaged to compare with the values on

labels. The TAC we determined and TAC certified were analyzed for systematic deviations by paired Students t test. Forensic data analysis: Aggregated test results were acquired from the “seed-to-sale” databases of several states. We analyzed these datasets with R Studio and/or Python. We authenticated datasets using individual certificates of analysis, sales data, and ancillary data provided by individual testing labs. RESULTS/DISCUSSION Testing off-the-shelf: The three labs returned values of TAC that were similar (average standard deviation = 1.8 wt%), and each set of 15 TAC values were statistically indistinguishable (p = 0.926). We determined mean values of TAC less than those certified by an average of 5.9 wt%; for 7 of 15 products, we measured TAC under the label by >20%. The likelihood that natural variance provided these results paired with those certified is less than one percent (p-value = 7.5*10-6). Out of ten samples we submitted for microbiological testing, two exceeded action limits for fungi and bacteria. One contained 65000 CFU/g yeast and mold, and 15000 CFU/g bile-tolerant bacteria; species included Klebsiella michiganensis bacteria and potentially pathogenic species of yeast. A second contained 150000 CFU/g aerobic bacteria. Our demonstration of microbiological contaminants and discrepant label values of TAC reveals hidden risks of cannabis from dispensaries. Degradation of cannabinoids cannot explain discrepancies in TAC, because we did not observe the elevated levels of cannabinol that accompany aging.6 By examining this ‘mislabeling’ critically, and consulting with industry professionals at multiple Independent Testing Labs and elsewhere, we determined that some portion of the discrepancy in values of THCA and other principal cannabinoids might be attributed to differences in water content of the cannabis, because this factor is not controlled in the potency testing of cannabis in MA. Nonrepresentative sampling of the cannabis batch at the time of its compliance testing (i.e., different quality buds in the compliance test than in the package) may also factor. Additional mechanisms for discrepant values in the compliance testing of cannabis have been articulated, many of them fraudulent.7 Forensic data analysis: We propose, and demonstrate briefly here, the application of two methods of data analysis for the testing results in seed-to-sale databases. While these analyses cannot unambiguously prove fraud on their own, they provide regulators and accrediting bodies with indications to investigate. We divided test results for Max THC by the lab which provided them and binned them to 1% ranged to construct the histograms in Figure 1. Figure 1 presents histograms from two labs, on the left, with a normal distribution of test results, on the right, a discontinuous distribution.

Figure 1. Frequency analysis of aggregated results of Max THC tests from two labs. On the left, a lab whose results form a continuous normal distribution, and on the right, a lab whose results form a discontinuous distribution. Blue bars denote values of Max THC below 20 wt%, and red bars denote values above 20%. We also tracked the average results of potency tests for individual producers over time, analyzing for changes in the testing lab providing the results. In some cases, test submitters began receiving tests from a new provider, with a simultaneous increase in the average potencies certified (Figure 2). Figure 2. Plots of average Max THC test results provided to individual testing customers. Circles denote average of all Max THC results in a given batch of test results for one of three selected customers. A change in the colors of circles denotes a change of lab. Horizontal bars denote averages of all the results displayed for the lab of the matching color. CONCLUSIONS We conclude that the nascent markets for cannabis in many states provide cannabis which often falls short of its label claims. We assert that the aphorism, “You don’t control what you don’t measure,” applies to many regulated cannabis markets. No testing is performed at the point of consumption, so no management is achieved of the condition of cannabis that is consumed. Regular shelf tests gave us useful feedback about the quality of products that the MA adult-use market provides. The results of these retests suggest that failures of quality assurance are common in Massachusetts. We would urge using different criteria than we did for selection of samples to include more products in future work. Aggregated testing data may be examined for anomalous distributions of the resulting values. Such analyses may be applied to cannabinoid levels, but also to numeric results of tests for contaminants with discrete action limits.

Testing and other market data may also be examined for evidence that some lab companies compete with others on results, such as a producer changing their lab vendor while simultaneously increasing the average levels of cannabinoids certified for their cannabis, or lowering the fail rates of QC tests. ACKNOWLEDGEMENTS MCR Labs, Framingham, MA REFERENCES 1. Vandrey R, Raber JC, Raber ME, Douglass B, Miller C, Bonn-Miller MO. Cannabinoid Dose and Label Accuracy in Edible Medical Cannabis Products. JAMA. 2015;313(24):2491. doi:10.1001/jama.2015.6613 2. Zoorob MJ. The frequency distribution of reported THC concentrations of legal cannabis flower products increases discontinuously around the 20% THC threshold in Nevada and Washington state. Journal of Cannabis Research. 2021;3(1)doi:10.1186/s42238-021-00064-2 3. Paulson E, Swider J, Eisenberg Z. The Inflated THC crisis plaguing California cannabis. Cannabis Industry Journal: Cannabis Industry Journal; 2022. 4. Jikomes N. Weed buyer beware: THC inflation is getting out of hand. Leafly. Accessed 2/14/2023, 2023. https://www.leafly.com/news/science-tech/marijuana-thc-inflation-is- getting-out-of-hand 5. Sagar KA, Gruber SA. Marijuana matters: reviewing the impact of marijuana on cognition, brain structure and function, & exploring policy implications and barriers to research. International Review of Psychiatry. 2018;30(3):251-267. doi:10.1080/09540261.2018.1460334 6. Ross SA, ElSohly MA. CBN and D9-THC concentration ratio as an indicator of the age of stored mrijuana samples. United Nations Office on Drugs and Crime blog. 12/1/1999, 1999. https://www.unodc.org/unodc/en/data-and-analysis/bulletin/bulletin_1997-01- 01_1_page008.html#5 7. Toth J. Smoke and Mirrors: 10 Ways The Cannabis Industry Can Manipulate THC Percentages. Eye On Cannabis blog. 4/30/2023, 2023. https://www.eyeoncannabis.org/blog/smoke-and-mirrors-10-ways-the-cannabis-industry- can-manipulate-thc-percentages

“Trajectory-Irrational to Rational”: Online COMPENDIUM 50/5* of Microbial Testing Regulations Speeds Identification of Issues in Developing a Consensus Set of Required Tests (*50 States/5 Territories) Authors: Sherman Hom, Dhanasri Nair, Dhanashree Pawar, Shravani Chobhe; Medicinal Genomics Corporation, Beverly, MA ABSTRACT SUMMARY Microbial contaminants in cannabis and hemp products pose a health hazard to consumers if these contaminants are human pathogens. Because of its FDA scheduling as a Class I agent, one single regulatory guideline for cannabis product microbial testing does not exist. As of August 2023, 39 States, 4 US territories, & the District of Columbia have legalized medical cannabis, and 24 States, 2 US territories, & the D.C. have legalized adult use cannabis. Thirty-eight (38) States, 2 US territories, and the D.C. have adopted microbial testing regulations. The number of required microbial tests ranged from 1 to 10 per jurisdiction. Action levels for” total count” tests differed by several ORDERS OF MAGNITUDE from State to State or Territory. In contrast, the action levels for Genus & species tests were either “<1 cfu/g” or “none detected”. From 2019 to 2023, the percentage of jurisdictions that require flower testing for specific microbial pathogens was found to have increased. INTRODUCTION Microbial contaminants in cannabis and hemp products pose a health hazard to consumers if these contaminants are human pathogens [1-10]. At particular risk are patients who are immunocompromised. These patients include those using cannabis to treat the nausea/vomiting and/or pain associated with cancer or advanced AIDS (acquired IMMUNO-DEFICIENCY syndrome). Because of its FDA scheduling as a Class I agent, one single regulatory guideline for microbial testing of cannabis does not exist. The resultant plethora of State regulations poses a public health risk. Medicinal Genomics Corporation (MGC) has monitored Cannabis testing regulations as the industry evolves. We here report a study of the changes in microbial testing regulations between 2019 and early 2023. TEST METHOD/OVERVIEW The most current cannabis testing regulations were reviewed [11]. For each jurisdiction with legal cannabis production, required microbial tests with the corresponding action level were recorded for each product type (flowers, concentrates, edibles, and transdermal products). Comparative analyses were conducted within & among each product type. Finally, data in the 2023 Compendium was compared to that from a 2019 Compendium of cannabis testing regulations. RESULTS/DISCUSSION As of August 2023, 39 States & 4 US territories have legalized medical cannabis, and 24 States & 2 US territories have legalized adult use cannabis. The District of Columbia has legalized both medical and adult use. Thus, the total number of jurisdictions that have instituted some form of legalization is 43. Of these 43 jurisdictions, 38 states, 2 US territories, and the District of Columbia have adopted microbial testing regulations. Two of these states have distinct sets of microbial testing rules for medical and adult use cannabis programs. The number of required microbial tests ranged from 1 to 10 per jurisdiction. Action levels for” total count” tests differed by several ORDERS OF MAGNITUDE from State to State or Territory. In contrast, the action levels for Genus & species tests were almost always “<1 cfu/g”

and “none detected”. For the different cannabis product types, most of the jurisdictions have unique combinations of required microbial tests, and the lists of required tests vary greatly. From 2019 to 2023, the percentage of jurisdictions that require flower testing for specific microbial pathogens was found to have increased (e.g., Aspergillus fumigatus by 30% to 56%). Jurisdictions requiring “total count tests” have decreased (e.g., Total Bile-Tolerant Gram-Negative Bacteria by 62% to 32%). The number of jurisdictions that have unique test combinations has slightly increased. For cannabis to be regarded seriously as medicine, it must be shown to be safe, even in immunocompromised patients. In the “post-Covid19” era, young adults, older adults, and the elderly all fear “long Covid” or “breakthrough Covid” symptoms. The adult recreational consumer also requires basic safety. Bacteria, yeasts, and molds that cause an immune response in humans–i.e., clinically-relevant human pathogenic microbes—should not be present in cannabis products. The healthcare system would be further burdened by these potentially confounding pathogens due to increasing use of legal cannabis and decreased black-market products. To that end, safety testing regulations for cannabis flower–a unique type of plant material, used by the medical or recreational consumer in a large variety of dosage forms–cannot rationally be written for legislative review in a cut-and-paste format from other industries or jurisdictions. Microbial testing regulations for cannabis need to be configured, first, with knowledge of cannabis-specific science–which considers cannabis’ unique properties and the specific human pathogens it may host–and secondly, the regulations need to be pertinent to the specific microbial organisms that may cause harm or cause disease in humans. Utilization of these two major guidelines will help speed arrival of the common-sense endpoint of a rationally consistent, coherent, and comprehensive set of testing regulations. Publication of such a set of “GMP” testing regulations may help many States and Countries consider legalization; and will improve the safety of cannabis on the open market. In this way, public health will also be served. CONCLUSIONS In the absence of federal legalization, more jurisdictions will modify & adopt their own lists of microbial testing regulations. If current trends prevail, specific pathogen detection by Genus and species will increase, and “total” counts of microbial populations will decrease. At this moment, however, required test combinations for cannabis products remain extremely diverse. A national survey of flower microbiomes could identify any human pathogens of concern that may inhabit cannabis flowers. That, plus the already-known list of human pathogenic microbes that may end up on crops through the necessary processes of fertilization, watering, and human handling could produce a rational list of potential microbial contaminants. A single consensus set of universally recognized human pathogens as the rational targets for testing regulations has yet to emerge, but the current trend, as shown by this survey study, is in this direction. Continuing research and education are needed. ACKNOWLEDGEMENTS Anne Conyers-Hom, MD, PharmD and Frank Priscaro - Abstract critical review and editing REFERENCES 1. M.J. Chusid, J.A. Gelfand, C. Nutter, and A.S. Fauci, Letter: Pulmonary aspergillosis, inhalation of contaminated marijuana smoke, chronic granulomatous disease. Annals of Internal Medicine 82(5), 682-683 (1975). https://pubmed.ncbi.nlm.nih.gov/1094876/ 2. R. Llamas, D.R. Hart, and N.S. Schneider, Allergic bronchopulmonary aspergillosis associated with smoking moldy marihuana. Chest 73 (6), 871-872 (1978). https://journal.chestnet.org/article/S0012-3692(16)61841-X/pdf

3. S. Sutton, B.L. Lum, and F.M. Torti, Possible risk of invasive aspergillosis with marijuana use during chemotherapy for small cell lung cancer. Drug Intelligence & Clinical Pharmacy 20(4), 289–291 (1986). 4. R. Hamadeh, A. Ardehali, R.M. Locksley, and M.K. York, Fatal Aspergillosis associated with smoking contaminated marijuana in a marrow transplant recipient. Chest 94(2), 432–433 (1988). 5. W.H. Marks, L. Florence, J. Lieberman, P. Chapman, D. Howard, and P. Roberts, et. al., Successfully treated invasive pulmonary aspergillosis associated with smoking marijuana in a renal transplant recipient. Transplantation 61(12), 1771–1774 (1996). 6. M. Szyper-Kravitz, R. Lang, Y. Manor, and M. Lahav, Early invasive pulmonary aspergillosis in a Leukemia patient linked to Aspergillus contaminated marijuana smoking. Leukemia & Lymphoma 42(6), 1433–1437 (2001). 7. R. Ruchlemer, M. Amit-Kohn, and D. Raveh, et. al., Inhaled medicinal cannabis and the immunocompromised patient. Support Care Cancer 23(3), 819–822 (2015). 8. D.W. Cescon, A.V. Page, S. Richardson, M.J. Moore, S. Boerner, and W.L., Invasive pulmonary aspergillosis with marijuana use in a man with colorectal cancer. Journal of Clinical Oncology. 26(13), 2214–2215 (2008). 9. A. Bal, A.N. Agarwal, A. Das, S. Vikas, and S.C. Varma, Chronic necrotizing pulmonary aspergillosis in a marijuana addict: a new cause of amyloidosis. Pathology 42(2), 197–200 (2010). 10. Y. Gargani, P. Bishop, and D.W. Denning, Too many moldy joints - marijuana and chronic pulmonary aspergillosis. Mediterranean Journal of Hematology and Infectious Diseases 3, 2035- 3006. Open Journal System (2011). 11. Cannabis Microbial Testing Regulations by State https://medicinalgenomics.com/resource/cannabis-microbial-testing-regulations-by-state

Residual Natural Constituents in Industrial Hemp Authors: Frederic Vallier1, Ted Haney1,2 1Federation of International Hemp Organizations (FIHO), Brussels, Belgium; 2Canadian Hemp Trade Alliance (CHTA), Calgary, AB, Canada ABSTRACT SUMMARY Plants produce inherent toxins, which are naturally occurring substances that a plant produces as a means to protect itself from environmental stressors. Most human food, livestock feed, and therapeutic plants are all considered safe to consume although their levels of inherent toxins can vary depending on the variety of the plant, the growing conditions, and other factors. Hemp also generates inherent toxins, but the lack of commonly accepted regulations leads authorities to be much more restrictive for hemp than for other plants because of the confusion that still exists between non-intoxicating hemp and high-THC Cannabis. Cannabinoids, including THC, are natural constituents of the Cannabis sativa L. plant and its derivatives, and not to be regarded as contaminants. For the residual contents of natural constituents to be limited in food we propose the term “Residual Natural Constituents”. The Federation of International Hemp Organizations (FIHO), representing producers and manufacturers, proposes that a commonly accepted regulation for Residual Natural Constituents be applied to hemp products like other processed food products. Regulations should be fair, compared to other products, and coordinated between the different regulators in order to facilitate the sale and trade of food and feed products derived from Hemp. INTRODUCTION Industrial hemp (hemp) is the common English name for a multi-use Cannabis sativa L. plant with low tetrahydrocannabinol (THC) concentration in the tops. While hemp is indigenous to Eurasia and Africa, humans have propagated it worldwide over many centuries. In fact, humankind’s global relationship with hemp spans millennia; with production and trade in a large array of hemp products, used for many purposes across time, continents, and civilizations. Indeed, few agricultural crops provide such a wide range of uses as hemp while offering abundant opportunities for climate change mitigation, biodiversity, and resiliency, adaptation, smart agriculture, prosperous resilient rural economies, and increased domestic self-reliance in food security and manufacturing self-reliance. With its seeds, hemp provides protein- and energy-rich grains for food and feed, as well as nutritionally valuable oil. With its flowering tops, hemp provides wellness products that are in high demand by consumers worldwide, the (early harvested) leaves provide an excellent source of plant protein and traditional ingredient for herbal tea infusions. With its stalks, hemp provides many wide-ranging applications in insulation, building materials, textiles, paper and packaging, renewable energy, advanced bio-composites, graphene, bioplastics, and more. With its deep and complex roots, hemp contributes to soil health (organic matter content, and nutrient holding capacity), water quality (soil holding capacity and reduced runoff), and soil carbon sequestration (1 hectare (2.5 acres) of hemp reckoned to absorb 8 to 22 tons of CO2 a year). Humans have been consuming hemp for centuries using rudimentary extraction methods (manual threshing). Thus we can deduce that human beings have been exposed to higher levels

of THC in food compared to the current levels used by the industry today. Modern food manufacturers have been producing and trading hemp food for decades using the most advanced cleaning processes and are able to provide consumers with a safe product. Nowadays THC levels are measured in “ppm” which means parts per millions! Indeed, no health-related intoxication from hemp seeds has ever been recorded. THC as inherent toxins Plants produce inherent toxins, which are naturally occurring substances that the plant produces as a means to protect itself from environmental stressors. Most human food, livestock feed, and therapeutic plants, such as cereal grains1, grass seeds2, roots and tubers3, vegetables4, pseudocarps5, fruits6, tree nuts7, and ground nuts8, are all considered safe to consume although their levels of inherent toxins can vary depending on the variety of the plant, the growing conditions, and other factors. Hemp also generates inherent toxins but the lack of commonly accepted regulations leads authorities to be much more restrictive for hemp than for other plants because of the confusion that still exists between non-intoxicating hemp and high-THC Cannabis. Cannabinoids, including THC, are natural constituents of the Cannabis sativa L. plant and its derivatives, and not to be regarded as contaminants. For the residual contents of natural constituents to be limited in food we propose the term “residual natural constituents”. By this, we define the residual level present in hemp food after certain (industrial) processing measures. The sole exception to this definition is the extraction of concentrated and isolated cannabinoids from the flowering tops. Food derived from hemp contains traces of cannabinoids, which must be considered to be a residual natural constituent of hemp. TEST METHOD/OVERVIEW The Federation of International Hemp Organizations (FIHO), representing producers and manufacturers, is conducting/will conduct a comparative review of all available/published HBGV, LOAEL, and NOAEL values related to the oral consumption or topical use of products derived from Industrial Hemp, including issuing requests for the scientific data used to set these levels. Resources such as UN Commission on Narcotic Drugs, World Health Expert Committee on Drug Dependence (ECDD), US Food and Drug Administration, National Institutes of Health (NIH), Health Canada, Food Standards Australia and New Zealand (FSANZ), Swiss Federal Office of Public Health (SFOPH), European Union EFSA, among others, will be consulted to consolidate and compare this information. The FIHO will then propose that a commonly accepted regulation for Residual Natural Constituents be applied to hemp products like other processed food products. The pathway to setting these levels may be similar to other established forms of setting Maximum Residue Limits, such as those observed by Health Canada or the OECD for setting Pesticide MRLs. A further goal would be to have this practice eventually adopted by the FAO Codex Alimentarius. 1 wheat, oats, and barley 2 rye, sorghum, corn, sugar cane, and lemongrass 3 beets, cassava, carrots, onions, potatoes, radishes, sweet potatoes, and yams 4 broccoli, Brussels sprouts, cabbage, cauliflower, cucumber, lettuce, peas, pumpkin, spinach, mustard, zucchini 5 apples, pineapple, pears, and strawberries 6 blueberries, Kiwi fruit, mangos, plums, and tomatoes 7 almonds, Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, and walnuts 8 peanuts, bambara, and hausa

RESULTS/DISCUSSION Regulations should be fair, compared to other products, and coordinated between the different regulators in order to facilitate the sale and trade of food and feed products derived from Hemp. The overall uncertainty factor (or safety factor) applied to the LOAEL in most countries for deriving a Health Based Guidance Value (HBGV) for THC is set much too high for such a substance of relatively low toxicity, compared to other toxic substances of concern in food or consumer products, such as alcohol, caffeine (from cocoa, kola, coffee, and green tea), nicotine (from tobacco, potatoes, tomatoes and eggplants), glycoalkaloid/solanine (from potatoes and tomatoes, and, opium and morphine (from poppy seeds). There is no scientific evidence that sub-psychoactive levels of THC in foods have any significant negative effects on human health. As such, it is constraining for a growing industry to be faced with overburdensome regulations on Residual Natural Constituents. CONCLUSIONS The potential of Hemp is not to be contested; however, the development of the Hemp industry will depend on the capacity to overcome burdens and unnecessary regulatory processes. With this open discussion, the FIHO wants to change the image of regulators towards Hemp to unlock its development and show that Hemp is a natural product with no more if not less Residual Natural Constituent than most other crops. ACKNOWLEDGEMENTS

1. Keanan Stone, Co-chair Research and Standard Committee of the FIHO (Canada)

2. Lorenza Romanese, Director General of the European Industrial Hemp Association (Belgium)

3. Daniel Kruse, President of the European Industrial Hemp Association (Germany)

4. Ted Haney, Board Chair of the Federation of International Hemp Organizations (Canada)

5. Patrick Atagi, Chair of the National Industrial Hemp Council of America (USA)

6. Charles Kovess, Chair of the Australian Industrial Hemp Alliance (Australia)

7. Rick Fox, Co-chair of Policy and Regulations Committee of FIHO (USA)

8. Michael Bronstein, CEO, American Trade Association for Cannabis and Hemp (USA)

9. Hunter Buffington, Co-chair of the Sustainability Committee of FIHO (USA)

10. TerryGrajczyk,Co-chairoftheSustainabilityCommitteeofFIHO(Canada) REFERENCES

• Federal Agency for Toxic Substances and Disease Registry (ATSDR) (https://www.atsdr.cdc.gov)

• European Food Safety Authority (EFSA) (https://www.efsa.europa.eu/en)

• UN Conference on Trade And Development (UNCTAD) (https://unctad.org)

• US Drugs and Food Administration (USDA) (https://www.fda.gov)

• UN Commission on Narcotic Drugs (UNCND) https://www.unodc.org

• World Health Expert Committee on Drug Dependence (ECDD)

• https://www.who.int/groups/who-expert-committee-on-drug-dependence

• Canadian National Institutes of Health (CNIH), Health Canada, https://www.cnih.ca

• Food Standards Australia and New Zealand (FSANZ) https://www.foodstandards.gov.au

• Ministry For Primary Industries, Government of New Zealand https://www.mpi.govt.nz

• Swiss Federal Office of Public Health (SFOPH) https://www.bag.admin.ch

• Organization for Economic Co-operation and Development (OECD)

• https://www.oecd.org

In Search of a Multiresidue Method for Pesticides Control in Hemp, Psychoactive and Recreational Cannabis Produced in Uruguay Authors: Mónica Pereira1,2, Florencia Tissot2, Ignacio Machado2 1Graduate Program in Chemistry, Facultad de Química , Universidad de la República, Montevideo, Uruguay; 2Analytical Chemistry, Facultad de Química, Universidad de la República, Montevideo, Uruguay ABSTRACT SUMMARY In recent years, some countries, including Uruguay, have legalized the use of medicinal and recreational cannabis. This has produced an increase in the production and consumption of cannabis and derived products, which is why the need to ensure the quality and safety of this type of resource. In this context, the aim of this work is to develop and validate a method for pesticide residues determination in cannabis flowers, to achieve adequate quality control and safety of these products. The scope of pesticides was selected according to the formulations allowed in these crops in Uruguay and based on the list of the Ph. Eur. (2021), since Europe is the main export destination. Determinations were carried out by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Sample preparation was optimized by modifying a citrate QuEChERS protocol (Anastassiades et al., 2003). The developed method showed good performance for the target analytes in terms of recoveries, precision, limits of quantitation and matrix effect. This tool is expected to expand the analytical capacity of the group, with application and impact at the level of research in a topic in continuous growth. It will also be of great help to producers, who will have the necessary information to be able to take corrective measures, in case of finding levels above the maximum permitted levels, either in crops or by-products. The method was applied to real samples of hemp and psychoactive cannabis (not recreational), revealing the presence of pesticides such as chlorpyrifos, diazinon, fipronil, tebuconazole, fludioxonil and ciprodinil. In this regard, the establishment of maximum residues limits (MRLs) for contaminants in cannabis in Uruguay seems to be an urgent necessity, especially for those pesticides registered for its use and commercialization, although its use is not destined for cannabis crops. Keywords: cannabis, pesticides, QuEChERS, multiresidue analysis. INTRODUCTION The monitoring of recreational cannabis, both psychoactive and hemp, includes different actions with the aim of ensuring the maximum safety to its consumers. It should cover all stages from production to consumption. In addition, safety is essential for public health and a very relevant factor in marketing. The implementation of good agricultural practices and extreme aftercare in the following stages of industrialization are crucial to ensure the quality and safety of these products. But it is not always enough, since plants are exposed to natural factors that constitute a silent danger to consumers (Dryburgh et al., 2018). Plants are susceptible to be attacked by insects, fungi, and diseases throughout its cultivation, being pesticides commonly used to prevent it. If waiting times or applications are not properly carried out, residues can remain both in plants and derived products. This is important for extracts, where not only cannabinoids are concentrated but also pesticide residues. In this regard, a given pesticide residue may be undetectable in the plant material but exceed the recommended limit in the extract (Craven et al., 2019). Uruguay does not have its own regulation of maximum limits of pesticides, which poses a risk for the user, and even more considering that products based on cannabis sativa extract are prescribed for children. Only for recreational cannabis, IRCCA (Institute for Regulation and Control of Cannabis) established for crops that regulates, the maximum limits for cyprodinil, fludioxonil, dimethoate and abamectin. Thus, the aim of this work was to develop a simple and

rapid method, employing and analytical techniques accessible in our laboratory, able to cover the needs of both local and international specifications for pesticide residues monitoring. Also, to apply the validated method as a routine and reference method for control agencies. TEST METHOD/OVERVIEW Samples: Samples used for validation were purchased at authorized recreational cannabis sale locations. A total of 50 real samples provided by different producers were analyzed. Extraction method: Briefly, 2g of dried homogenized sample was weighed in a 50 mL polypropylene tube. Afterwards, 10 mL of water were added and samples vortexed for 10 min for hydration. Later, 10 mL of 0.1% acetic acid (Carlo Erba, France) in acetonitrile (Carlo Erba, France) were added. After that, 1g of sodium chloride (Carlo Erba, France), 1g trisodium citrate dihydrate (Baker, Mexico), 0.5 g disodium hydrogen citrate sesqui-hydrate (Acros, USA) and 4g of magnesium sulfate (Carlo Erba, France) were added and samples shaken for 5 min in multiples vortexes. Tubes were centrifuged for 5 min at 4000 rpm, and a 6 mL portion (previously neutralized with ammonium hydroxide) was transferred to a 15 mL polypropylene conical tube containing 900 mg of anhydrous magnesium sulfate, 300 mg primary secondary amine (Macherey Nagel, Germany) and 200 mg graphite carbon black (Sigma Aldrich, USA). The extract was filtered through a 0.22 μm PVDF (Whatman Uniflo, USA) filter and injected into the LC–MS/MS system. Instrumentation: A triple quadrupole MS/MS SCIEX 3500 coupled to SCIEX Exion LC system was used. An Agilent Technologies Zorbax Eclipse XDB-C18 (100 mm×2.1mm, 3.5μm) was used for LC separation and the column oven temperature was set at 30°C. The mobile phase consisted of (A) 0.1% formic acid and 5 mM ammonium formate (Carlo Erba, France) in water and (B) 0.01% formic acid and 5 mM ammonium formate (Carlo Erba, France) in methanol (Carlo Erba, France). The following elution program was used: started at 5 % B (held for 1 min), increased to 98 % B over 5 min (held for 7 min), and finally the percentage of B was decreased to 5 % over 3 min (held for 3 min), making a total run of 17 min. Injection volume was 10 μL and flow rate was fixed at a 0.3 mL min−1. RESULTS/DISCUSSION The aim of this work was to develop a simple and rapid clean-up method, for the simultaneous determination of 66 pesticides residues in cannabis flowers, to be applied for the monitoring by liquid chromatography, including a group of pesticides that are commonly determined by gas chromatography. The focus of the work was on the optimization of the extraction process while using a 3 ^ 3 factorial design. Thus, QuEChERS extraction time, amount of PSA and amount of GCB were evaluated, achieving a quantitative extraction (Pérez-Parada et al., 2016). According to the SANTE/11312/2021 document, recoveries % (R%) using a suitable matrixmatched calibration must be in the range 70% - 120%, after spiking with appropriate volumes of spiking mix solution at 0.1 mg kg-1 (expressed as fresh weight). For the target contaminants, an R% at the lower end of this range is adequate because typically these contaminants are present at trace and ultratrace levels. Tandem MS detection was performed using the multiple reaction monitoring (MRM) mode. The optimal MRM conditions for each analyte were optimized using direct infusion in the ESI+ (Electrospray ionization) mode. Repeatability, trueness, limit of detection (LD) and limit of quantification (LQ), linearity and dynamic range, and matrix effect were the main figures of merit evaluated, according to the recommendations of SANTE/11312/2021. For recovery and precision studies, samples were spiked with appropriate volumes of spiking mix solution at 0.001, 0.05 and 0.1 mg kg-1 (expressed fresh weight). Repeatability was evaluated as % RSD (relative standard deviation), obtaining values between 6% and 20%. Meanwhile, trueness was evaluated as % R, obtaining

values in the range 70-120%. The LQ was set at 0.01 mg kg-1 (expressed as fresh weight) based on the MRLs of the European Pharmacopoeia, and it was experimentally verified after evaluating the %R at this level. The LD was set at 0.005 mg kg-1 (expressed as fresh weight), half the LQ according to the recommendations of CRLs 20/1/2010. Good linearity was observed for all pesticides with determination coefficients (R2 ) greater than 0.98. Individual residuals were also studied, and their random distribution verified. Thus, the lineal dynamic range was 0.01-0.25 mg kg-1, although for those with high sensitivity the range was 0.01-0.10 mg kg-1 (expressed as fresh weight). All determinations were performed by matrix-matched calibration, due to a negative matrix effect observed (Pérez-Parada et al., 2019). Method performance was in accordance with the recommendations of the validation guide used as a reference, suggesting that the proposed method is suitable for the purpose of the work. After validation, this method was applied for the monitoring of commercial flower samples. Obtained results were below the LD, but diazinon, tebuconazole, fipronil and chlorpyrifos were quantified in 6 of the samples. These pesticides are registered and allowed in Uruguay. Furthermore, the flower samples that presented quantifiable levels were outdoor plantations. For this reason, these residues might have been incorporated into the plant through the air, animals, soil, or water since it is not common its use in cannabis crops. The global pharmaceutical industry is very demanding, so ensuring quality and meeting international standards is a must. In this regard, it is necessary to develop analytical methods able to contemplate the greatest number of pesticides possible, including the ones regulated at the national level. Only cyprodinil, fludioxonil, abamectin and dimethoate have a MRLs set by IRCCA for recreational cannabis. The establishment of MRLs in cannabis in Uruguay seems to be necessary and important soon, according to the positive findings in some of the analyzed samples. The validated method applied for the constant monitoring of pesticides is of outmost importance to help producers, who will have the necessary information to take corrective measures, in case of finding levels above the maximum allowed limits, either in crops or derived products. CONCLUSIONS An analytical method based on a clean-up and extraction by citrate QuEChERS method with acetonitrile, was developed for the determination of 66 pesticides including organochlorides, organophosphorus, carbamates and pyrethrins in hemp and recreational flowers. The results of the validation showed that the method can be considered as a reference standard method for cannabis safety monitoring. In addition, it was possible to apply this alternative method for all the studied contaminants by liquid chromatography. Cannabis flower samples from Uruguay were analyzed using the developed method, and in general the concentrations of studied pesticides were far below the corresponding maximum limits allowed as indicated by Ph. Eur. (2019). REFERENCES Anastassiades, M., Lehotay, S.J., Stajnbaher, D., Schenck, F.J. (2003). Fast and easy multiresidue method employing acetonitrile extraction/partitioning and ‘dispersive solid phase extraction’ for the determination of pesticide residues in produce, Journal of AOAC International, 86(2), 412– 431. Brown, A. K., Xia, Z., Bulloch, P., Idowu, I., Francisco, O., Stetefeld, J., Stout, J., Zimmer, J.,Marvin, C., Letcher, R. J., & Tomy, G. (2019). Validated quantitative cannabis profiling for

Canadian regulatory compliance—Cannabinoids, aflatoxins, and terpenes. Analytica ChimicaActa,1088, 79-88. COMMUNITY REFERENCE LABORATORIES RESIDUES (CRLs) 20/1/2010. Guidelines for the validation of screening methods for residues of veterinary medicines. Craven, C.B., Wawryk, N., Jiang, P, Liu, Z., Li, X.F.(2019). Pesticides and trace elements in cannabis: Analytical and environmental challenges and opportunities, Journal of Environmental Sciences, 85, 82–93. European Commission–DG-SANTE (2021). Document SANTE/11312/2021. Analytical quality control and method validation procedures for pesticide residues analysis in food and feed. Supersedes guidance document SANTE/12682/2019. Implemented by 01/01/2022. European Pharmacopoeia (Ph. Eur.) (2021). Herbal drugs. Instituto de Regulación y Control del Cannabis (IRCCA) (2023) https://www.ircca.gub.uy/ Pérez-Parada, A., Alonso, B., Rodríguez, C., Besil, N., Cesio, V., Diana, L., Burgueño, A.,Bazzurro, P., Bojorge, A., Gerez, N., Heinzen, H. (2016). Evaluation of three multiresidue methods for the determination of pesticides in marijuana (Cannabis sativaL.) with liquid chromatography-tandem mass spectrometry, Chromatographia, 79, 1069–1083.

Agricultural Agents: How Laboratories Inability to Correctly Quantify Could be Putting Consumers at Risk Authors: Jini Curry Glaros, Wyatt Bergel, Derek Perkins; Modern Canna Labs, Lakeland, FL ABSTRACT SUMMARY Cannabis is an extremely complex matrix that has chemical properties which can make the quantitation of various analytes within the product difficult. As such, laboratories must develop methods that will account for the matrix effect and interferences that naturally occur in everyday samples. While most analyses can be impacted by matrix, agricultural agent testing is impacted the most. As such, laboratories must take the matrix and resulting interferences into consideration when processing samples. If a laboratory wants to accurately identify and quantify compounds of interest, matrix calibrations, the use of internal standards, and robust method development are essential. This presentation will provide data and evidence regarding the impact that a matrix can have on agricultural agent results and why the lack of matrix calibrations can be detrimental to the accurate identification and quantitation of the analytes. Additionally, tools for identifying matrix interferences and how to combat these issues will be discussed in further detail. Cannabis laboratories must focus on producing data that is accurate, reproducible, and legally defensible to help ensure consumer safety. Consumption, whether through inhalation or ingestion, of toxic agricultural agents can be extremely dangerous to the health of consumers and it is the responsibility of the laboratory to ensure that all products on the market are safe. As such, this research and its supporting evidence play a critical role in the cannabis industry and the laboratories ability to protect the consumer. INTRODUCTION The accurate identification and quantitation of contaminants is imperative to ensure consumer safety. As such, laboratories must ensure that the data they are producing is accurate, reproducible, and legally defensible. Cannabis testing can be difficult, especially for analytes such as agricultural agents, where laboratories are expected to detect contaminants at extremely low limits in a sample with heavy matrix interference. One of the biggest hurdles that cannabis laboratories face is ensuring that the methodology being used can properly identify and quantify agricultural agents. While some laboratories have implemented the use of matrix calibrations for agricultural agent testing, others are still preparing calibrations in solvent and quantifying samples using this technique. If a laboratory does not create a matrix specific calibration that is similar to the matrix of the samples being analyzed, there is a significant possibility that agricultural agents are not being identified or quantified accurately, and in most cases, this inaccurate quantitation results in values that are skewed low. As a result, cannabis users may be consuming product that is not actually safe for human consumption. TEST METHOD/OVERVIEW To provide a clearer understanding of the impact that the cannabis matrix and the interferences that exist have on the results when testing for agricultural agents, the laboratory implemented the following experiment and utilized an Agilent 6470 LC-MS/MS for the data acquisition and analysis. Various cannabis samples (flower, derivative, and edible products) were extracted via the standard laboratory protocol prior to being analyzed for agricultural agents via LC-MS/MS. The laboratory prepared a solvent calibration for the samples, along with blended matrix calibrations for each sample type (flower, derivative, and edible matrices). The laboratory then separated an aliquot of sample after extraction and spiked it with a known amount of all agricultural agents being measured. The samples and their spiked counterparts were then analyzed against the solvent calibration and the corresponding matrix calibration to determine the recovery for each

analyte. The information was tracked in the laboratory information management system (LIMS) so that the recovery percentages for each sample could be calculated for both the solvent calibration and the matrix calibration. Additionally, for problematic analytes which do not recover well for a solvent or matrix calibration, the laboratory established a protocol that can be used to accurately quantify the analyte(s) of concern. Through the use of standard addition, which involves creating a calibration for the analyte of interest from the sample in question, the laboratory is able to extrapolate the true concentration of the contaminant. RESULTS/DISCUSSION By conducting this series of experiments, the laboratory was able to prove that the use of matrix calibrations are imperative for the proper identification and quantification of agricultural agents. In the experiments, the laboratory utilized a passing recovery range of 80-120% for the analytes being spiked into the samples. In doing this, the laboratory was able to show that blended matrix calibrations reduce the number of recovery failures for flower by 36.1%, for derivatives by 61.4%, and for edibles by 76.5%. Furthermore, the reduction of low failures was even more impactful as the results showed that the laboratory was able to reduce these by 69.4%, 75.0%, and 83.9% for flower, derivatives, and edibles, respectively. When conducting this type of testing, it is better to have failures high rather than low, as low failures may indicate that laboratories are under quantifying analytes that are present, thus potentially putting consumers as risk. When recovery failures do exist, either high or low, the laboratory was able to confirm that the use of standard addition will correct any issues with the quantification of the analyte(s). By creating a five-point calibration curve, with two values below the expected concentration of the analyte, one at approximately the same concentration, and two values above the expected concentration, the laboratory was able to accurately quantitate the analyte and remove any matrix interferences that existed. As such, it is important that laboratories utilize quality control, so that they are regularly spiking unknown samples (MS/MSD), to ensure that the matrix is not leading to the suppression or enhancement of the ions used to quantify the result. CONCLUSIONS When cannabis laboratories are testing for agricultural agents, it is imperative that matrix calibrations are used. Matrix calibrations should be a blend of multiple different samples that have a variety of interferences and should be similar in complexity to the samples being analyzed. Based on the data obtained, laboratories should be creating a matrix calibration for each sample type that is being analyzed by the laboratory. Since matrix calibrations are generated from blended samples that are free of all analytes of interest, laboratories can divide samples into the following categories – flower, derivative, and edibles – and have a calibration for each. Additionally, if and when interferences are present that impact the laboratories’ ability to accurately identify and quantify an analyte, even after a matrix calibration, the laboratory should use standard addition to ensure that the contaminants are being quantified properly. If laboratories are not currently implementing matrix calibrations and the use of standard addition for the identification and quantitation of agricultural agents in samples, there is a high possibility that laboratories are not properly reporting results for this category of contaminants. As such, the laboratories’ inability to accurately identify and quantify these analytes pose a risk to consumer safety. Cannabis laboratories and regulators must implement matrix calibrations as a requirement to ensure that contaminated product does not make it to market. The laboratories primary concern should be consumer safety and the ability to detect and quantify agricultural agents accurately must be a top priority.

ACKNOWLEDGEMENTS This work was undertaken through internal funding at Modern Canna Labs. REFERENCES 1 Stone, P., Hitchcock, J., Roy, JF., & Deckers, C. (2020). Determination of Pesticides and Mycotoxins as Defined by California State Recreational Cannabis Regulations. Agilent Technologies Application Note. 2 Hajšlová, J., & Zrostlıková Jitka. (2003). Matrix Effects in (Ultra) Trace Analysis of Pesticide ́ Residues in Food and Biotic Matrices. Journal of Chromatography A, 1000(1-2), 181–197. 3 Giacinti, G., Raynaud, C., Capblancq, S., & Simon, V. (2016). Matrix-Matching as an Improvement Strategy for the Detection of Pesticide Residues. Journal of Food Science, 81(5).

Enhancing Confidence in Microbial Contaminant Testing Through the Addition of Viability Test Solutions Authors: Patrick M. Bird, Maria McIntyre, John Mills, Ronald Johnson, Adam Joelsson, Ashley Brown; bioMérieux, Hazelwood, MO ABSTRACT SUMMARY Recently published studies from industry and data from state regulatory associations have indicated that fungal contaminants, specifically related to pathogenic Aspergillus species, are routinely isolated from cannabis plants. Industry estimates indicate that ~20% of cannabis flower batches fail testing requirements, with most due to total fungal contamination or the presence of pathogenic Aspergillus. Cultivators struggle with fungal contamination found in grow facilities and throughout the supply chain; resulting in a reliance on post-harvest remediation steps designed to reduce or eliminate microbial flora present in their products. These steps can be effective; however, depending on both the type of remediation utilized as well as the application of the remediation step, these processes may reduce the bioburden in the product but not eliminate the presence of bacterial or fungal DNA. This poses a problem to industry as testing for pathogen microorganisms relies on highly sensitive methods, such as PCR, that can detect the presence of this DNA even when the organisms are no longer viable. To resolve this issue, technology providers have developed solutions that eliminate the presence of DNA from non-viable organisms in cannabis products prior to analysis by PCR. The GENE- UP® PRO Viability Kit was validated as a solution to remove DNA present in the environment as well as from non-viable cells prior to detection with the GENE-UP® Aspergillus PRO test method. A matrix study in dried hemp flower was performed according to AOAC Appendix J microbiology validation guidelines and AOAC SMPR 2019.001 to demonstrate the fitness for purpose of the PRO Viability Kit with Aspergillus PRO for detection of viable Aspergillus in cannabis. Results of the validation study demonstrated that the IS Viability Kit was successful in removing non-viable DNA from the matrix prior to detection by PCR and the method was granted AOAC Performance Tested MethodSM certification. INTRODUCTION Aspergillus includes many species, about 40 of which have been implicated in human or animal infections (1). Aspergillosis is the general term used to describe the various infections (allergic sinusitis, chronic pulmonary, invasive) caused by these species (2). Most cases of aspergillosis are caused by Aspergillus fumigatus, followed by Aspergillus flavus and Aspergillus niger (1). While less common, aspergillosis infections caused by A. terreus have the highest mortality rate of all Aspergillus species (1). These infections mainly occur in immunocompromised individuals and are the most severe forms of infections caused by Aspergillus species (3). As states have legalized cannabis for medicinal and/or adult-use, testing data indicates that the presence of pathogenic Aspergillus is widespread in cannabis markets (4). TEST METHOD/OVERVIEW A method validation study was conducted on dry hemp flower inoculated with Aspergillus niger utilizing the GENE-UP® PRO Viability Kit as an upfront DNA clean-up procedure prior to analysis by the GENE-UP® Aspergillus PRO assay. Testing was performed using two test portion sizes (10 g and 1 g) in compliance with AOAC SMPR 2019.001 guidelines (5) and meet the needs of state regulatory agencies. Three levels of contamination were evaluated 20 replicates at a low-level of approximately 2.0 cfu/test portion, 5 replicates at a high-level of approximately 20 cfu/test portion, and 5 replicates at an non-inoculated control level of 0 cfu/test portion. Test portions were inoculated, stabilized and evaluated according to AOAC Appendix J validation guidelines (6). Test portions for both 1 and 10 g were evaluated with and without the

viability PCR reagent using both a 24 h and 48 h testing protocol. Regardless of presumptive results, all test portions were confirmed by plating onto DRBC agar plates after 48 h of enrichment. Macroscopic and microscopic evaluation of isolated colonies were performed. RESULTS/DISCUSSION Results obtained in the study, at both 24 h and 48 h, met the requirements from AOAC Appendix J guidelines for fractional positive results (25 – 75 % positive results at the low level of contamination). Probability of detection (POD) analysis indicated that results between presumptive PCR analysis and culturally confirmed results were equivalent. Test portions analyzed without the PRO Viability kit produced 4 results that were not able to be confirmed. When analyzed using the PRO Viability kit, results aligned with culture confirmation indicating that the initial results were detection of DNA from non-viable cells in the sample. The data from these studies, within their statistical uncertainty, support the product claims of the PRO Viability kit as a DNA removal preparation step for samples analyzed by the GENE-UP Aspergillus PRO. CONCLUSIONS The GENE-UP® Aspergillus PRO is a real-time PCR assay that is AOAC PTM certified and has been adopted for use across the US. With the inclusion of remediation techniques to eliminate pathogenic Aspergillus species from cannabis products post-harvest, DNA clean-up techniques are a necessity for the industry to have accurate results on remediated cannabis flower. With the validation of the PRO Viability kit as a DNA removal step, the cannabis industry now has a protocol for removing DNA from intact non-viable cells as well as from the environment prior to analysis by PCR. ACKNOWLEDGEMENTS All testing was performed by paid representatives of bioMérieux. REFERENCES (1) Health Canada (2011). Pathogen Safety Data Sheets: Infectious Substances – Aspergillus spp. https://www.canada.ca/en/public-health/services/laboratory-biosafety- biosecurity/pathogen-safety-data-sheets-risk-assessment/aspergillus.html (Accessed March 2023) (2) Centers for Disease Control and Prevention (2023). About Aspergillus https://www.cdc.gov/fungal/diseases/aspergillosis/definition.html (Accessed March 2023) (3) Llamas, R., et al. (1978). Allergic bronchopulmonary aspergillosis associated with smoking moldy marihuana. Chest, 73(6), 871-872. (4) Thompson, G.R., Tuscano, J.M., Dennis, M., Singapuri A., Libertini S., Gaudino R., et al. (2017). A microbiome assessment of medical marijuana. Clinical Microbiology and Infection. 23(4):269-70. (5) AOAC International SMPR 2019.001, Standard Method Performance Requirements for Detection of Aspergillus in Cannabis and Cannabis Products. http://www.eoma.aoac.org/SMPR/upload/116/SMPR%202019_001.pdf (accessed March 2023) (6) Official Methods of Analysis (2019) 21st Ed., Appendix J: AOAC INTERNATIONAL. Rockville, MD, http://www.eoma.aoac.org/app_j.pdf (Accessed March 2023)

A Regulatory Perspective on Challenges in Cannabis Testing and Consumer Safety Moderator: Gillian Schauer, PhD, MPH, Executive Director, Cannabis Regulators Association (CANNRA) Speakers: Lori Dodson, MS, MT(ASCP), Senior Advisor, Maryland Cannabis Administration John Kaba, Ph.D., Environmental Manager-Laboratories, Office of Medical Marijuana Use, Florida Department of Health Heather Krug, M.S., Regulatory Programs Branch Chief, Colorado State Public Health Laboratory, Division of Disease Control and Public Health Response (DCPHR), Colorado Department of Public Health and Environment (CDPHE) ABSTRACT SUMMARY While cannabis remains illegal at the federal level, adult use and medical markets are becoming increasingly common across the United States. As of May 2023, medical cannabis is legal across thirty-eight states, three territories, and the District of Columbia. Additionally, twenty-two states and the District of Columbia have legal adult use markets. Revenue projections from various models show cannabis revenue topping $30 billion in 2022 and projected to hit $56.9 billion in 2028. With such a lucrative and dynamic market comes remarkable innovation, but that innovation inevitably introduces significant challenges for regulators regarding testing standardization and product safety. Innovation outpaces science, and science out paces regulation. With a vast array of cannabis products readily available to consumers across the country, it is critical that standardized product testing models become available and are consistently used as a tool to ensure product safety. Standardized product testing models would not only provide a level playing field for industry, but they would also provide regulators across the country with tools to protect public health from common contaminants in cannabis and cannabis products. In this session, regulators from three states discuss the on-the-ground challenges of staying on pace with innovation while implementing cannabis testing regimes to protect consumer safety.