Microbiological Attributes of Powdered Cannabis

Friday, July 31, 2015

Introduction

Although marijuana is classified as a Schedule I drug “having no medical use, with a high potential for abuse” by the U.S. Federal Drug Enforcement Administration, Western States including Colorado, Alaska, Oregon and Washington have legalized the sale of powdered cannabis for both recreational and medical use. As powdered cannabis has the potential for microbial contamination, during cultivation, harvesting, drying, storage and distribution with fecal pathogens, molds, especially Aspergillus fumigatus, and aflatoxin, attention must be given to its production and appropriate microbiological test procedures and specifications established that reflect user risk.

Setting microbiological specifications is complicated by having two broad user groups, namely recreational and medical users and that powdered cannabis is most commonly smoked but also may be used as an ingredient in baked goods and further processed to an extract. In medical use, smoking cannabis has been used to control weight loss associated HIV-AIDS, prevent nausea associated with chemotherapy, and alleviate pain associated with a range of illnesses. These patient populations will be more susceptible to microbial infection than recreational users. With respect to mode of administration, the microbiological quality requirement will be different for cannabis that is smoked or eaten. As many medicinal cannabis users have severely compromised immune systems, medical cannabis distributed by licensed producers in the Netherlands and Canada is irradiated for control of the bioburden.

Does the scientific literature report concerns with the microbial contamination of cannabis? There are multiple reports of immuno-compromised individuals receiving chemo-therapy and corticosteroids, solid organs and stem cell transplant recipients and HIV/AIDS patients contracting chronic pulmonary aspergillosis (CPA) associated with medical or recreational use of cannabis (Chusid et al, 1975; Llamas et al, 1978; Sutton et al, 1986; Hamadeh et al, 1988; Denning et al, 1991; Mark et al, 1996; Szyper-Kravitz et al, 2001; Cescon et al, 2008; Bal et al, 2010; Gargani et al, 2011). In addition to CPA that has a high mortality rate, if not treated early, chronic use of smoked cannabis is associated with allergic broncho-pulmonary aspergillus (ABPA) (Kagen et al, 1983).

Cannabis may be contaminated with other human pathogens during its cultivation, processing and distribution. Outbreaks of hepatitis B (Cates and Warren, 1975), hepatitis A (Alexander, 1987) and salmonellosis (Taylor et al, 1981) have been reported.

Published surveys of the microbial populations of powdered cannabis in peer-reviewed journals are few. Verweiji et al (2000) reported higher mold numbers using Sabouraud Dextrose Agar on cannabis compared to tobacco (200-300 versus 104-107 cfu/g) with the predominant fungi Penicillium spp, Aspergillus fumigatus and A. flavus. They reported that cigarette smoke was negative for mold. Other studies have shown that smoke from both burning tobacco and marijuana cigarettes contain fungal spores (Kagen et al, 1983; Pauly and Paszkiewicz, 2011). In an evaluation of the quality of medicinal-grade and coffee shop-purchased cannabis Hazekamp (2006) reported that irradiated medicinal-grade cannabis contained less than 10 cfu/g enterobacteria and Gram-negative bacteria (Bile-tolerant, Gram-negative bacterial count) and less than 100 cfu/g molds and aerobic bacteria (Total Aerobic Microbial Count) while ten sample of coffee shop cannabis ranged from less than 10 to 80,000 cfu/g bile-tolerant, Gram-negative bacterial count and 120 to 480,000 cfu/g Total Aerobic Microbial Count. The contaminants from one of the ten samples were identified as the bacterium E. coli and molds of the genera Penicillium, Cladosporium and Aspergillus.

How common is aspergillosis? As summarized by the Centers for Disease Control and Prevention (CDC) because aspergillosis is not reportable in the United States, the number of cases is difficult to determine. ABPA is a pulmonary disorder caused by immunologic reactions to antigens released by A. fumigatus, colonizing the airways of individuals with asthma and cystic fibrosis. Published studies cited in the CDC report suggests that ABPA affects between 1 and 15% of cystic fibrosis patients and 2.5% of adults who have asthma, an estimated 5 million people worldwide of which 400,000 have CPA (Stevens et al, 2003; Denning et al, 2013). Although ABPA is debilitating, it is not life-threatening, but invasive aspergillosis is less common and can to high mortality rates in immuno-compromised patients, especially among solid organ transplant recipients.

Fungal Microflora

What is the fungal microflora of the Cannabis sativa plant? They are the normal epiphytic and endophytic fungi on the plants that should have no impact on user safety. Furthermore C. sativa is susceptible to fungal pathogens with grey mold Botrytis cinerea, hemp canker Sclerotinia sclerotiorum, and leaf spot Septoria spp. being the most important (McPartland, 1996). More important to this discussion are so-called storage molds including Rhizopus nigricans, Mucor hiemalis, Penicillin chrysogenum, Aspergillus flacus and Aspergillus fumigatus. These molds will persist but not grow in dried powdered cannabis with a low water content (activity). The minimumwater activity for growth for these representative molds is found in Table 1.

Table 1. Minimum Water Activity for Growth for Common Storage Molds (After Cundell and Fontana, 2009)

Rapid drying of cannabis plant material from 60-80% to 6-12% moist content at temperatures below 40°C will prevent mold growth without degrading cannabinoids. The water activity will be reduced from 0.8-0.9, that readily supports the growth of the mold Aspergillus flavus stored at temperatures above 25°C, to 0.4-0.5 (Kulshrestha et al, 2008). Modeling the effect of water activity and temperature on the growth rate and aflatoxin production by A. flavus on harvested rice demonstrated an optimum growth temperature of 30°C with growth above 0.84 with higher toxin production at higher water activities (Mousa et al, 2011). Clearly water activity (content) is a critical physical attribute for the protection of powdered cannabis from storage molds. A review of the moisture sorption isotherms of spices, in absence of those for cannabis, indicates that unprotected exposure of powdered cannabis to humidity exceeding 80% will promote water adsorption and result in mold growth. Storing powdered cannabis in paper or jute bags is a bad idea, as it will not protect the product from elevated humidity.

Test Methods and Specifications

Although some testing laboratories have adopted test methods found in USP <61> and <62> for drug products and USP <2021> and <2022> for dietary supplements and the microbiological quality requirements for dried or powdered botanical found in USP <2023> there is no well established microbiological specification for powdered cannabis. Other standard-setting organizations including the American Herbal Products Association (AHPA), the European Pharmacopeia (Ph. Eur.), NSF Standards/American National Standards Institute (Dietary Supplements) and the World Health Organization (WHO) have published microbial limits for finished botanical products (Table 2). The 2013 American Herbal Pharmacopoeia Monograph – Cannabis Inflorescence and Leaf Table 9 recommended the microbial standards contained in USP <2023> with the exception that they required an absence of pathogenic strains of E. coli in 1 g.

Table 2. Microbial Limits for Finished Botanical Products (cfu/g)

The problem with adopting a microbiological specification for a finished herbal product for powdered cannabis is that this specification does not adequately address the critical quality attributes of an inhaled product. Based on a risk assessment, the largest concerns are fecal pathogens such as hepatitis A, Salmonella spp. and enteropathogenic E. coli, molds especially members of the genus Aspergillus and mycotoxin aflatoxin. Fecal pathogens are most likely to cause infection through handling powdered cannabis while Aspergillus spores and aflatoxin may contaminate smoke from burning cannabis in joints or water pipes and be drawn deep into the lungs. This suggests that the critical microbiological quality attributes for powdered cannabis should be the absence of E. coli as an indicator of recent fecal contamination, absence of Salmonella spp., the fungal count and perhaps aflatoxin levels as an indicator of appropriate post-harvest processing and drying. Perhaps an absence of Aspergillus spp. requirement would be more reflective of user risk than absence of Clostridium spp., S. aureus or Shigella spp. requirements.

The quality control specifications developed by Health Canada and the Dutch Office of medicinal Cannabis (OMC) are compared to the USP <1111> and <2013> recommendations (Table 3). Note: the USP cannabis monograph was withdrawn in 1941.

Table 3. Quality Control Specifications for Dried Marijuana (Cannabis)
Table 4. Different USP Microbiological Requirements for a Herbal Product for Oral Use and a Inhalation Drug Product (cfu/g)

The microbiological quality requirements for inhalation drug products as found in USP <1111> is stringent in terms of microbial counts and absence of specified microorganisms and would probably not be met by powdered cannabis useless it was irradiated (Table 4). Furthermore, although P. aeruginosa and S. aureus may be responsible for lower respiratory tract infections, they are unlikely to survive in powdered cannabis. However, based on the recently published foodborne illness source attribution estimates from the US Interagency Food Safety Analytics Collaboration (IFSAC) Project (2015) the number of outbreaks and the estimated number of outbreak associated illness attributed to seeded vegetables, which may most resemble cannabis, during 1990-2012 time period was 34 and 4001 respectively for Salmonella and none for E. coli O157. This suggests with powdered cannabis, Salmonella illnesses would be more of a risk than E. coli illnesses.

One strategy adopted by a prominent cannabis testing laboratory that may have some merit is to establish total aerobic microbial counts one log lower for smoked recreational versus edible cannabis and a further log lower for medicinal cannabis and for total combined yeast and mold counts one log lower for medicinal versus recreational cannabis (Table 5).

Table 5. Microbial Limits Strategy for Different Classifications of Powdered Cannabis (cfu/g)

Perhaps a more questionable marketplace-driven strategy is assigning quality levels Gold, Silver and Bronze and pricing accordingly (Table 6).

Table 6. Gold, Silver and Bronze Quality Designations and their Microbial Specifications

Recommendations

This author believes that the USP <2023> powdered herbal product microbiological quality requirements are not stringent enough for medicinal cannabis smoked by user populations that include the immuno-compromised. In the absence of bioburden-reduction steps such as irradiation for medical-grade cannabis, a possible microbiological quality requirement may be TAMC NMT 103 CFU/g,TCYMC NMT 102 CFU/g, and Absence of E. coli and Salmonella in 10 g. The critical unanswered question is whether this TCYMC limit would control the Aspergillus fumigatus content in powdered cannabis and if the addition of an absence of A. fumigatus and/or aflatoxin specification should be added for medicinal cannabis. Additional microbiological testing surveys of marketed powdered cannabis published in peer-reviewed journals would be informative in specification setting.

A strong argument can be made that the most valuable critical quality attribute to prevent fungal contamination of powdered cannabis may be water content (activity). Data on the moisture sorption isotherm for dried powdered cannabis would be useful in specification setting.

References

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  2. Bal, A., A.N. Agarwal, A. Das, S. Vikas and S.C. Varma. Chronic necrotizing pulmonary aspergillosis in a marijuana addict: a new cause of amyloidis. Pathology 2100; 42: 197-200
  3. Cates, W., and J.W. Warren. Hepatitis B in Nurenburg, Germany. JAMA 1975; 234: 930-934
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  6. Cundell, A.M. and A.J. Fontana. Appendix D in Water Activity Applications in the Pharmaceutical Industry. 2009: 276-277 DHI/PDA
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  9. Gargani, Y., P. Bishop and D. W. Denning. Too many moldy joints - Marijuana and chronic pulmonary aspergillosis. Mediter. J. Hematol. Infect. Dis. 2011; 3:e2011005 Open Journal System
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  11. Hazekamp, A. An evaluation of the quality of medicinal grade cannabis in the Netherlands. Cannabinoids 2006; 1(1):1-9
  12. Interagency Food Safety Analytics Collaboration (IFSAC) Project Foodborne illness source attribution estimates for Salmonella, Escherichia coli O157, Listeria monocytogenes and Campylobacter using outbreak surveillance data. February 2015
  13. Kagen, S.L., V.P. Kurup, P.G. Sohnle, and J.N. Fink. Marijuana smoking and fungal sensitization. J. Allergy Clin. Immunol. 1983; 71: 389-393
  14. Kulshrestha, R., C.P. Gupta, G. Shukla, M.G. Kunda, S.P. Bhatnagar and C.K. Katiyar. The effect of water activity and storage temperature on the growth of Aspergillus flavus in medicinal herbs. Planta Med. 2008; 74(10): 1308-1315
  15. Llamas, R., D.R. Hart and N.S. Schneider. Allergic bronchopulmonary aspergillosis associated with smoking moldy marijuana Chest 1978; 73:871-872
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  18. Pauly, J.L. and G. Paszkiewicz. Cigarette smoke, bacteria, mold, microbial toxins and chronic lung inflammation. J. Oncol. Vol. 2011, article ID 819129, 13 pages
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Author Biography

Dr. Tony Cundell works as a consulting microbiologist supporting the pharmaceutical industry. Prior to November 2013 he worked for Merck Research Laboratories in Summit, New Jersey, as the Senior Principal Scientist in early phase drug development. Earlier in his career, Tony Cundell worked at a director level in Quality Control and Product Development organizations at the New York Blood Center, Lederle Laboratories, Wyeth Pharmaceuticals and Schering-Plough.

He is a member of the 2015-2020 U.S. P Microbiology Committee of Experts. In June, 2009, he co-edited with Anthony Fontana a book entitled Water Activity Applications in the Pharmaceutical Industry and contributed two chapters to the book.

Tony Cundell has a Ph.D. in Microbiology from the Lincoln University, New Zealand.

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