Producing uncontaminated Cannabis on a commercial scale is nearly impossible, and largely unprecedented. Random tests of marijuana sold on the street suggest that as much as 40% has unsafe levels of mould, pests, insect droppings, chemical pesticides, and other potentially harmful adulterants.
With two product recalls within the first 30 days of the opening of the Canadian commercial Cannabis production market, the nuances of Health Canada’s justifiably stringent Quality Assurance (QA) guidelines are revealing their technicality and rigour. Patients and Physicians have spoken, and only producers who can stand behind Cannabis completely free of chemical pesticides, mould, pests, and hostile remediation activities such as irradiation will be recognized as top-level producers. Why is purity such a critical part of a Licensed Producer’s commitment to quality?
The QA Practices of a modern commercial Cannabis cultivator must be highly specialized, technical, and methodical. Details matter. For the more technically inclined, and in the interest of transparency, we at Tantalus Labs want to take this opportunity to educate the processes of prevention, detection, and natural remediation that we utilize to ensure patients can rely on pure and high caliber Cannabis from us and other producers at the forefront of Quality Assurance technology.
Cannabis is generally assumed by the medical community to be safe for human consumption. However, the product has been found to be contaminated by a large number of microbial organisms such as fungi or moulds, bacteria, plant viruses and protozoa. Unfortunately, these microbial contaminants, often originating in soil or derived from manure, can thrive under the same environmental conditions where the Cannabis is cultivated.
Many of these bacterial and mould species, along with their products (mycotoxins), may result in allergic reactions and diseases and can even cause life threatening infections in patients; especially those with compromised immune systems. Today, patients who require medicinal Cannabis have the right to insist on medicine that is free of all contaminants. If Cannabis carries mould, pests, insect droppings, chemical pesticides, and other potentially harmful adulterants, its risk profile for consumption expands greatly. If the case can be made that Cannabis has therapeutic potential, it must be stressed that purity and lack of contaminants are contingent to that potential. More conventional products for human consumption that one might buy in a pharmacy or grocery store have been run through a battery of sample tests to ensure their quality, and showcase the potency of any active ingredients clearly on the label. This is unfortunately not the case with marijuana purchased on the street.
While a large range of the harmless bacteria and fungi that form the naturally occurring microflora of Cannabis plants are innocuous, poor cultivation and production practices can cause further but preventable microbial contamination, resulting in risk of harm for patients.
Proliferation of contaminating microorganisms may result from improper planting density or irrigation practices, inappropriate crop rotation practices, lack of field sanitation such as destruction of crop residues, disinfection of tools, boots or greenhouse benches and equipment, the use of feces as manure, contaminated water, and poor worker hygiene during harvesting and processing of the plant product.
Also failure to control the temperature and moisture levels during drying, processing, transportation and storage after harvesting can further increase the risk of contamination. Microbial contamination may also occur through handling by personnel who are infected with pathogenic bacteria during harvest/collection, post-harvest processing and the manufacturing process, as well as risk of livestock and bird contact. The following lists a few of the microbial contaminants that may be found in medicinal Cannabis, especially that which is grown in cultivation environments that do not minimize mould growth risk. The following may adversely affect patients:
Aspergillus: One of the more potentially deadly moulds to humans. Some species produce large amounts of aflatoxin, which is both a toxin (mycotoxin) and a carcinogen. Exposure can cause allergic reactions and/or infections, especially in immune compromised patients.
Alternaria: A plant pathogen and allergen to humans. Exposure can cause hay fever and may lead to asthma. Exposure can also cause infections in immune compromised patients.
Cladosporium: Is a plant pathogen that can affect Cannabis size, yield and quality. This is the group that contains the most common moulds but generates no mycotoxins. Over exposure can cause asthma attacks and/or pneumonia in some patients.
Peniccillium: Exposure can cause severe allergic reactions in patients who are sensitive to the antibiotic Penicillin.
Yeasts: A group of microorganisms that are often found in Cannabis. Some are pathogenic and/or allergenic and should be avoided by immune compromised patients.
Escherichia Coli (E. coli): A gram negative bacteria that lives in the mammalian gastrointestinal tract. Most strains are harmless, however, several can cause food poisoning in humans.
Botrytis: A necrotrophic fungus that affects many plant species. Also known as grey mould or bud rot, it can result in respiratory allergic reactions.
Although many of these contaminants are completely destroyed when Cannabis is combusted or smoked, mould and bacterial contamination are now becoming more of a concern as patients turn to vaporization and edible products as alternative ways to ingest Cannabinoids. Even handling mouldy product can contaminate the skin and transfer to food or membrane, for instance when a contaminated individual rubs their eyes.
To address these concerns, producers have resorted to third party Quality Assurance, sending Cannabis to labs to be tested for bacteria, fungus, and mould. If a product has been found to have unacceptable levels of fungus or bacteria, producers have used gamma radiation to reduce or eliminate the contamination. The irradiated Cannabis is then sent back for retesting. It is important to note that this transfer and travel time removes the Cannabis from its controlled and Quality Assurance environment, further increasing risk of contamination.
Also, many patients are becoming concerned about the use of radiation to treat products for ingestion, as well as the fact that it is not a risk-free mechanism for eradicating mould and other adulterants. Although it can kill the viable mould and possibly the mould spores, it will not impact the aflatoxins, many of which are carcinogenic. It is also known that radiation can destroy the essential oils, called terpenes, which are naturally produced by the Cannabis plant and can play an important synergistic role in therapeutic response.
As such, Tantalus Labs believes that the best practice is to minimize contamination preemptively by adopting strict cultivation practices and procedures, where the Cannabis plants are grown in tightly controlled environments, in which water and air flow are stirctly filtered and carefully monitored. As well, our environment allows risk of animal and bird contact to be largely eliminated.
Throughout the process, the plants are closely monitored for contamination, and the final product determined contaminant-free through in-house testing prior to third party validation and release. The greenhouses and testing facilities are operated and controlled under a quality assurance plan using standard operating procedures and implementing best practices guidelines approaching current good manufacturing practice (cGMP) as defined in the Canadian Food and Drug Act.
Tantalus Labs will, therefore, test for E. Coli, salmonella, total aerobic bacteria, yeast, mould and coliforms etc. to ensure the product is completely safe from microbial contamination. Since there are currently no accepted standards for safe levels of fungi or bacteria in Cannabis, all release limits will be set at levels far below standards for other products meant for human consumption such as dietary supplements and tobacco set by organizations such as Health Canada, World Health Organization (WHO), United States Pharmacopeia (UPA), European Pharmacopeia (EP) and the American Herbal Products Association (AHPA).
Methods for determining numerical levels of moulds and bacteria have been established for over 40 years by organizations such as the USP and the National Science Foundation (NSF). Further identification of pathogens utilizes modern techniques such as microscopic inspection, polymerase chain reaction, enzyme linked immunoassays and chromatography of microbial toxins. All these techniques are used by Tantalus Pharmaceuticals to ensure a contaminant free product.
a) Microbial enumeration: Samples are initially tested for total amount of microbial contamination or density to verify the sample is safe for human consumption. Methods for microbial enumeration are laid out by the U.S. Pharmacopeia in USP 2021 and USP 61, and Tantalus Labs follows this strategy_._ Simply, this method involves plating out ground plant samples on agar plates at various dilutions and with replicates. After incubation over several days, colonies, if any, can be counted and colony forming units or CFU per gram can be determined for the original sample.
b) Identification: After estimation of microbial density, if colonies have formed it may be necessary to identify the detected microorganisms. Macroscopic and microscopic examination of colonies from the agar plates can often give a reasonably reliable initial identification. However, if it is necessary to quantify and produce a more rigorous characterization of the contaminants, then a more accurate method is required. In this case, Tantalus Labs uses quantitative (or real-time) Polymerase Chain-Reaction (qPCR) to rapidly identify the contaminants and amounts present in a given sample (e.g. species of fungi such as scopulariopsis, rhizopus, botrytis, clostridia, powdery mildew, as well as bacteria such as listeria, E. coli etc.). qPCR avoids the labor intensive and lengthy traditional plating methods (which involve plating, culturing and counting along with specific identity tests).
The technique is considerably more sensitive than the plating method and can detect as low as 50 CFU in under 5 hours as opposed to 5 days for plating based assays which often requires multiple platings to detect very low contamination levels. Also qPCR allows the detection of moulds and bacteria that cannot be cultured on agar. Furthermore, the analysis allows remedial action to be taken rapidly, which could be too late if using the slower plating method. qPCR is based on the polymerase chain reaction that utilizes DNA polymerase enzymes and primers to replicate and amplify small amounts of contaminant DNA in a sample.
The primers are short sequences of DNA that bind to certain points on the contaminant DNA and can be designed to attach to sequences that are specific to the contaminating species. Amplified DNA is then cut into shorter strands with restriction enzymes (enzymes that bind to, and cut, DNA at specific locations), which can then be separated by gel electrophoresis. Presence of a band on a gel can indicate a positive test for a particular species and the presence of that species in the sample. qPCR is and advanced form of PCR and allows the high throughput identification and quantification of multiple species of contaminants from a single sample. Primers are now available for many of the harmful moulds and bacteria since their DNA has been sequenced along with the genome of several Cannabis species. Ultimately tests such as these can be automated for very high throughputs, which will allow a more thorough screening of the live plants and finished product.
Detection of Mycotoxins
Mycotoxins, which include the aflatoxins produced by aspergillus species, are toxic metabolic products produced by fungi. Enzyme-linked immunosorbent assays or ELISAs are used to detect these mycotoxins using antibodies that bind specifically to the mycotoxin. The sample containing the toxin is bound to a well in a plate and the antibody, which is bound to an enzyme capable of catalyzing a reaction forming a colored product, is added. The plate is rinsed to remove unbound antibody. Finally, a substrate is added, and if antibody has been immobilized by binding to a target mycotoxin, detectable colored products are formed.
Unfortunately ELISA tests are specific to one mycotoxin and, therefore, a battery of tests are needed to detect all mycotoxins, which can be expensive and time consuming. To avoid this Tantalus Pharmaceuticals also uses high performance liquid chromatography (HPLC) to identify several mycotoxins in one sample. HPLC separates the components in a sample to identify and quantify each component. It relies on pumps to pass a pressurized liquid solvent containing the sample through a column filled with a solid adsorbent material. Each sample component including the toxins interacts differently with the adsorbent material, which affects their flow rate through the adsorbent material and leads to their separation as they flow out the column. The amounts of each component are then quantified by a fluorescent detector and compared to standard samples.
Microbial contaminants in Cannabis reccomendations from physicians can be harmful to patients, with particular risk for the immune compromised and critically ill. With the increasing use of edible forms of Cannabis, as well as vaporization, it has become essential for the patient to know that their Cannabis is free of all contaminants. To ensure this, Tantalus Labs has incorporated practices that minimize the potential of contamination through use of strictly controlled cultivation techniques in greenhouses that are operated under a quality assurance plan, using Standard Operating Procedures to monitor every step of the production process.
By avoiding contamination in the first place, these practices reduce the likelihood of using any form of irradiation to remove the contamination. Throughout the process, samples are tested for microbial contamination and, if detected, action is taken immediately to remedy the situation. The final product is also be tested in-house as well as third party validation before being declared contaminant-free and released to the patient. Tantalus Labs uses both traditional methods such as plating and ELISA as well as state-of-the art methods such as qPCR and HPLC to identify or declare the absence of the microbial contaminants.