Endotoxins and Mycotoxins

By Kevin She

During a recent conversation with a Cannabis/Medical Marijuana industry consultant, we again heard about another licensed operator whose facilities had become contaminated with mold and other microorganisms. Despite lengthy, costly, and downright Herculean efforts to remediate their facilities, the infestation had become endemic and could not be eradicated, and every crop that they produced could not pass inspection. The industry consultant then told us that the only recourse that company could take was to only produce Cannabis extracts.

My response was, “So they found a wholesale source of clean feedstock from another company to make their extracts with?”

Their response was, “…, uh, no.”

There are some factors here; endotoxins and mycotoxins are not tested for, the methods that are used to detect microbial contamination, the methods that Cannabis products and natural health products can be treated to pass microbial load inspection, and the long-term occupational safety of people who work in contaminated grow operations.

Firstly, what are toxins – and specifically what are endotoxins and mycotoxins?

Toxins are substances, typically small molecules produced by living organisms that can cause disease by interfering with enzymes or cell receptors or otherwise cause harm to the host. In contrast to venom, which is defined as a toxin produced by an animal for the purposes of causing harm, endotoxins are typically components of bacterial cells that our immune system recognizes and reacts to; usually appropriately, but sometimes not (for example, food poisoning). Mycotoxins probably fall somewhere in between.

Endotoxins are components of bacteria that our “primitive”/”first-line-of-defense” innate immune system recognizes as danger signals, first proposed as Pathogen Associated Molecular Patterns (now called Damage Associated Molecular Patterns) by the wonderful Dr. Polly Matzinger. Endotoxins include bacterial cell wall components like lipopolysaccharide (LPS) and peptidoglycan; flagellin, a component of some bacteria’s locomotive flagellum; and even bacterial DNA, among others. While endotoxins can elicit very strong immune responses, these components typically did not evolve to cause harm to a host organism but rather the host organism has evolved to recognize these highly evolutionarily conserved small molecules. Virulence factors, on the other

hand, are evolutionary adaptations that some microbes have developed to actively harm their host organism in various ways to gain a survival advantage for themselves.

One of the major lessons taught in food industries is that you can’t get rid of endotoxins in contaminated food; cooking contaminated food – even carbonizing everything (Weller 2014) – will not get rid of endotoxins that cause food poisoning. Indeed, studies in cigarettes show that LPS from bacteria contaminating tobacco leaves not only survive the burning process (Pauly 2011) the LPS in smoke remains bioactive, contributes to chronic inflammation, and can be present in levels comparable to levels of LPS that causes byssinosis (Hasday 1999), a chronic inflammatory lung disease that affects cotton, hemp, and jute workers (Shi 2010, Lai & Christiani 2014).

Endotoxins are not part of the required testing panel for medical Cannabis in Canada. In the United States, only California specifies testing for Aflatoxins B1, B2, G1, G2, and Ochratoxin A. For natural health products regulated by the Canadian NNHPD; only certain raw materials have to be tested for LPS.

Mycotoxins are difficult to define in a few words. In general, mycotoxins are small molecules that are produced by filamentous fungi. However, they are not required for the growth or survival of the fungi under most conditions, and many of their pathological effects in humans and animals do not necessarily benefit the fungi directly. The structures of different mycotoxins are highly diverse as are their methods of action (Bennet & Klich2003).

Some of the most important mycotoxins associated with human and veterinary diseases include aflatoxins, citrinin, ergot alkaloids, fumonisins, ochratoxin A, patulin, trichothecenes, and zearalenone.

Aflatoxins are a particularly carcinogenic mycotoxin. The liver has enzymes that break down aflatoxins (and a wide variety of other secondary metabolite small molecules); however, the breakdown products intercalate (gets stuck inside strands of) DNA and causes genomic damage when the cell divides, leading to cancer where these breakdown products accumulate – in the liver. Mycotoxin toxicity from moldy grains has been recognized since antiquity (Richard 2007), and the ancient Romans were well aware of liver cancer (Papavramidou 2010). Recently, archeological excavation of an ancient Roman site consisting mostly of the remains of poor working-class Romans revealed that they died young (around 30) and had extraordinarily high incidences of bone cancer – possibly a result of metastatic liver cancer (livers are soft tissue and are not preserved whereas bones can survive millennia). Interestingly, similar excavations of much more affluent

citizens of Pompeii who could presumably afford non-moldy food were free from bone tumors. Aflatoxin remains an important risk even today; it is estimated that aflatoxins contaminate approximately 25% of agricultural products worldwide and continue to contribute to liver failure and liver cancer (Yard 2013).

Fumonisins are a diverse family of mycotoxins produced by various species of Fusarium, a mold that is extremely common in improperly operated Cannabis growing facilities, that disrupts lipid metabolism in animals. Additionally, Fusarium contamination has been implicated in esophageal cancer (Sydenham 1991, Peraica 1999), acute effects similar to severe food poisoning (Bhat 1997), and may cause neural tube defects (grossly defective brain development in fetuses) (Hendricks 1999a, Hendricks 1999b).

Aflatoxins, but not fumonisins, are required to be tested for in medical Cannabis under the Canadian MMPR program, but neither are required to be tested for in jurisdictions where medical and/or recreational Cannabis are legal in the United States. For natural health products regulated under the Canadian NNHPD, only certain raw materials – for example, roots like ginseng or valerian – require testing for aflatoxins.

Medicinal Cannabis in Canada, under the MMPR, and medicinal and recreational Cannabis in legal United States jurisdictions do require microbial load testing. Acceptable microbial loads mean no endotoxins or mycotoxins, right? Not so fast.

Microbial enumeration testing methods approved by the European Pharmacopoeia and the United States Pharmacopeia (and indeed, all standard methods used for all microbial enumeration) can only test for live and cultivable microorganisms. A sample to be tested is ground up and mixed in sterile water. A series of known dilutions of this mixture, along with positive and negative controls, are plated onto selective media (a mixture of nutrients that promote the growth of particular kinds of microorganisms and inhibits the growth of others, for example, there are media that promotes the growth of yeasts and molds but not bacteria, and there are media that promote the growth of bacteria but not yeasts and molds and there are media that is mostly specific for E. coli or mostly specific for Salmonella).

Live and viable microorganisms grow and divide and over the course of 1-3 days form “colonies” that can be counted by eye. The number is called CFU (colony forming units) and is calibrated by the mass of the material that was tested (usually 1 gram, although for some tests it has to be demonstrated that there are zero CFU per 5 or 20 grams of material tested).

There are ways around high microbial loads, though. For cannabis, there are commercial drying machines that quickly dry cannabis flowers at high temperatures (instead of curing the flowers more slowly in a controlled environment). The desiccation of the flowers and the high temperature can decrease viable microbial counts – but it doesn’t get rid of the microbes nor the endotoxins and mycotoxins that they produce and are still present.

Another common treatment is gamma irradiation – some Canadian MMPR licensed producers tout their use of gamma irradiation as a positive – where the material is exposed to a high powered gamma radiation source. The amount of gamma radiation that is used is calibrated to be sufficient to cause enough DNA damage that no cells remain viable (able to successfully divide). Like with high-temperature drying, although this treatment reduces viable microbial counts, it doesn’t get rid of the dead remains of the microbes nor the endotoxins and mycotoxins that are already present.

So, back to the reported company that couldn’t get rid of their mold problem.

The production of extracts (“waxes,” “oils”) concentrates the cannabinoids from Cannabis plant parts into a much smaller volume and discards most of the plant material. Aside from the “cold water method” (dry ice), most extraction/concentration processes use organic solvents.

Since the extraction process is non-conducive to microbial survival, microbial loads are not required to be tested for (but residual solvents are).

However, most endotoxins (including LPS) and most mycotoxins (including aflatoxins and fumonisins) are all very highly soluble in the same solvents that are used to concentrate cannabinoids from Cannabis plant material (Bennet & Klich 2003). As the cannabinoids are concentrated into waxes and oils, so are any endotoxins and mycotoxins that are present.

Endotoxins and mycotoxins are not required to be tested for in Cannabis extracts in the United States. Aflatoxins, but not fumonisins, are tested for under the Canadian MMPR program.

Buyers beware, and hope for your sake and for the health of the people who cultivated and prepared the product that the producer grows clean!