Multi-Mycotoxins in Milled Grain Products and Grain-based Foods – April 1, 2015 to March 31, 2018

Targeted surveys are used by the CFIA to focus its surveillance activities on areas of highest health risk. The information gained from these surveys provides support for the allocation and prioritization of the agency's activities to areas of greater concern. Originally started as a project under the Food Safety Action Plan (FSAP), targeted surveys have been embedded in the CFIA's regular surveillance activities since 2013. Targeted surveys are a valuable tool for generating information on certain hazards in foods, identifying and characterizing new and emerging hazards, informing trend analysis, prompting and refining health risk assessments, highlighting potential contamination issues, as well as assessing and promoting compliance with Canadian regulations.

Food safety is a shared responsibility. The CFIA works with federal, provincial, territorial and municipal governments and provides regulatory oversight of the food industry to promote safe handling of foods throughout the food production chain. The food industry and retail sectors in Canada are responsible for the food they produce and sell, while individual consumers are responsible for the safe handling of the food they have in their possession.

Mycotoxins are natural toxins released by moulds that can grow on crops in the field or after harvest^{Footnote 1}. These toxins are released by moulds which can grow on agricultural products, such as on cereals (e.g. wheat, oats, and corn), legumes, nuts and fruit. The type of agricultural product, insect damage, and the climatic conditions (temperature, humidity) during growth, processing, and storage are some factors that can influence the types and levels of mycotoxins present in the foods available at the retail level^{Footnote 2}. The human health effects are varied; ranging from gastrointestinal distress to cancer, the health effects depend on the type and level of mycotoxin in the food.

Research has shown that of the hundreds of mycotoxins associated with food, a small fraction has the potential to adversely affect human health and pose a global health concern^{Footnote 2}. The Codex Alimentarius Commission is an international body established by the United Nations' Food and Agriculture Organization and the World Health Organization to develop harmonized international food standards, guidelines, and codes of practice to protect the health of the consumers and to ensure fair practices in the food trade. Codex has published a Code of Practice to reduce and prevent mycotoxin contamination in cereals (e.g., wheat, corn, oats, and barley)^{Footnote 2}. This Code of Practice acknowledges that the complete elimination of mycotoxins from foods is not possible but it provides guidance on ways to control and manage the mycotoxin levels at the farm level and after harvest (for example, during processing, storage, and transport).

There are now more than 300 known mycotoxins of widely different chemical structures and differing modes of action – some target the kidney, liver, or immune system and some are carcinogenic. Common mycotoxins include aflatoxins, ochratoxin A, ergot alkaloids, fumonisins, trichothecenes (such as deoxynivalenol which is also known as vomitoxin) and zearalenone^{Footnote 1}.

Please see Appendix A for a list of the analytes included in the method. Please consult Appendix B for description of the health effects of the different myoctoxins.

A variety of domestic and imported milled grains (bran, flour, meal, starch, whole grains) and grain-based foods (baked goods, breads and bread products, cookies, baking mixes, crackers, pasta). The samples were collected over 3 fiscal years (2015-16 fiscal year – April 1, 2015 to March 31, 2016; 2016-17 fiscal year – April 1, 2016 to March 31, 2017; and 2017-18 fiscal year – April 1, 2017 to March 31, 2018). Samples of products were collected from local/regional retail locations located in 6 major cities across Canada. These cities encompassed 4 Canadian geographical areas:

• Atlantic (Halifax)
• Quebec (Montreal)
• Ontario (Toronto and Ottawa)
• West (Vancouver and Calgary)

The number of samples collected from these cities was in proportion to the relative population of the respective areas.

Samples were analyzed by an ISO/IEC 17025 accredited food testing laboratory under contract with the Government of Canada. See Appendix A for a list of the analytes included in the method. The results are based on the food products as sold and not necessarily as they would be consumed.

Health Canada has not established tolerances or standards for the majority of the mycotoxins in the grain products targeted in this survey. In 2009, Health Canada proposed maximum levels (ML) for OTA in a variety of foods. An ML of 3 ppb has been proposed for grains for direct consumption (barley, oats, rice and wheat) and derived cereal products (for example flour, bread, breakfast cereal), an ML of 7 ppb has been proposed for wheat bran and an ML of 0.5 ppb has been proposed for infant formulas and cereal-based foods^{Footnote 3}. These MLs as well as an industry guidance value for OTA in unprocessed cereal grains are still under consideration.

In the absence of applicable tolerances or standards, high levels of mycotoxins may be assessed by Health Canada's Bureau of Chemical Safety (BCS) on a case-by-case basis using the most current scientific data available.

Multi-mycotoxins

A total of 2244 samples were analyzed for the presence of mycotoxins. The products sampled were separated into 16 types of milled grain products (bran, flakes, flour, grains, grits, groats, meal) and 9 types of grain-based foods (baked goods, bread products, baking mixes, cookies, crackers, pasta, snacks). Mycotoxins were detected in 1132 samples (50%). A total of 22 of 25 different mycotoxins were detected in the product types sampled in this survey. Aflatoxin G2, 3-Ac-DON, and 15-Ac-DON were not detected in any of the samples. Table 2 illustrates the number of samples with detectable levels of the various family of compounds for all of the product types. Spelt-based foods (cookie) and oat-based foods (crackers, baking mixes) have the highest and lowest percentage of detectable mycotoxins, respectively.

As can be seen from Table 2, up to 6 mycotoxins out of a possible 25 compounds were detected per sample. These may consist of multiple compounds from the same toxin family (3 forms of aflatoxin or 3 forms of fumonisin) or discrete mycotoxins (for example, sterigmatocystin).

As can be seen in Table 3, deoxynivalenol was the most frequently detected toxin (887 or 40% samples). The least commonly detected mycotoxins were aflatoxin B2; aflatoxin G1, fusarenone-X, and neosilanol, which were each detected in only one sample each. The levels ranged from 0.40 ppb to 3900 ppb.

A number of studies have been published about impact of farming method (organic, conventional) on mycotoxin levels^{Footnote 4, Footnote 5, Footnote 6, Footnote 7, Footnote 8}. There is not a clear, consistent link between farming method and the prevalence or levels of mycotoxins. This is also the case in these targeted surveys. The milled grains and grain-based foods varied in the proportion of conventionally grown and organic products (based on label claims). The breakdown of results of multi-mycotoxin testing in conventionally grown and organic grain-based products is presented in Table 4. The percentage of organic products containing mycotoxins decreased in the order: kamut and spelt-based foods (100%) > amaranth (98%) > rye (87%) > quinoa (84%) > buckwheat (75%) > oat-based foods (50%) > millet (35%) > barley (27%) > mixed grains (25%) > corn and rice (18%) > wheat (14%) > spelt (10%) > mixed grain foods (7.7%) > arrowroot (5.3%) > rice-based foods (5.0%) > wheat-based foods (2.7%) > teff (2.1%)> corn-based foods, rye-based foods, sorghum and teff-based foods (0%). Of the 18 product types which included conventionally grown and organic products, 3 had similar detection levels (oat-based foods, rice, corn), 8 had higher detection rates in conventional products, and 7 were associated with higher detection rates in organic products.

There was also no clear trend observed with respect to number of mycotoxins per sample and growing method – 4 product types (barley, corn, millet, wheat) showed no difference, 6 had a higher number in organic products (amaranth, buckwheat, oat, quinoa, rye, and spelt) and 6 had a higher number in conventionally grown products (arrowroot, mixed grains, mixed grain foods, rice, teff, and wheat-based).

n/a = no samples fit this description so cannot report on prevalence of mycotoxins or on maximum number of mycotoxins per sample

In comparison to previous survey years^{Footnote 9}, the detection rates for mycotoxins in various types of grain-based foods were generally consistent, with the exception of sterigmatocystin, as noted in Table 4 below. This may be related to differences in product types, conditions during a specific growing year, source of the grains, and/or use of fungicides. Health Canada has not established maximum limits for any of the mycotoxins in products included in this survey, with the exception of OTA^{Footnote 3}. The compliance rate for grain based products for OTA (97.1 % in 2015-16 fiscal year, 99.8% in 2016-17 fiscal year and 97.5% in 2017-18 fiscal year was comparable to previous survey years (96.5%). As observed in previous surveys, DON was the most commonly observed mycotoxin.

Health Canada has determined that the levels of mycotoxins in the grain-based foods observed in the current survey are not expected to pose a concern to human health, therefore there were no recalls or follow up actions resulting from this survey.

The data associated with this report will be accessible on the Open Government Portal.

Appendix B

1 Aflatoxins

Aflatoxins are a family of naturally-occurring, toxic secondary metabolites produced by Aspergillus flavus and A. parasiticus fungi^{Footnote 10}. Aflatoxin-producing fungi may contaminate agricultural products (such as corn, nuts, spices, dried fruit) if grown, transported, stored, or processed under hot, humid conditions for prolonged periods of time, or with pest pressures resulting in bruising or cuts on the commodity^{Footnote 10, Footnote 11}. Drought pressure on corn is also a major risk factor for the occurrence of aflatoxins in the field^{Footnote 10, Footnote 11, Footnote 12}. Due to the cooler Canadian climate, domestically-grown agricultural commodities (and products) are less likely to contain aflatoxins than those imported from warmer climates. Aflatoxins are not destroyed by heating, cooking or most other processing methods^{Footnote 13}.

One aflatoxin form, aflatoxin B1, is among the most potent naturally-occurring liver carcinogens known^{Footnote 14}. The International Agency for Research on Cancer (IARC) classified aflatoxins to be carcinogenic to humans (Group 1 carcinogen)^{Footnote 15}. Chronic exposure to aflatoxins has also been associated with growth impairment in children living in developing countries where exposure to aflatoxins is relatively high. Aflatoxins have been shown to cause immune suppression in experimental animals^{Footnote 16, Footnote 17, Footnote 18, Footnote 19}. Short-term exposure to high levels of aflatoxins can cause illness in humans which is characterized by vomiting, abdominal pain, convulsions, coma and death. The illness is very rare in the developed world^{Footnote 20}. This study included aflatoxins B1, B2, G1 and G2.

2 Cyclopiazonic Acid

Cyclopiazonic acid (CPA) is produced by Penicillium cyclopium, Penicillium species (e.g. P. commune and P. camembertii), Aspergillus flavus and A. versicolor. Cyclopiazonic acid has been detected in corn, millet, peanuts, pulses, cheese, ham, sausage, hot dogs, tomato and milk^{Footnote 21}.

There is little information available regarding potential human health effects associated with CPA. However, it has been linked to 'Kodua' poisoning in India resulting from ingestion of contaminated millet seeds. The symptoms included sleepiness, tremors and giddiness which lasted 1-3 days, followed by complete recovery^{Footnote 22}. Experimental animal studies indicate that CPA is toxic only when ingested in high concentrations. Repeat exposure to high doses of CPA show a range of effects such as neurotoxicity, liver and kidney damage, weight loss, diarrhea, dehydration, convulsions and death in several different species^{Footnote 23}.

3 Ergot Alkaloids

Ergot alkaloids (EA) are formed by fungi of the Claviceps species, particularly C. purpurea. These fungi parasitize the seed heads of cereals, replacing individual grain kernels with discoloured fungal structures (dark purple or black) known as sclerotia or ergot bodies. The predominant ergot alkaloids present in ergot bodies are ergometrine, ergotamine, ergosine, ergocristine, ergocryptine and ergocornine (only ergosine, ergocristine and ergocryptine were successfully included in the current multi-mycotoxin method). The type and levels of these alkaloids in ergot bodies vary considerably depending on the fungal strain, the host species, the weather conditions and geographic region. Wet weather and soil favour the growth of ergot bodies. These bodies are harvested with the cereals and can thus lead to contamination of cereal based food and feed products with ergot alkaloids. The cleaning methods used during grain processing usually remove the ergot bodies from the grain^{Footnote 24}.

Long-term exposure to ergot alkaloids causes ergotism, also known as ergotoxicosis, ergot poisoning and Saint Anthony's Fire^{Footnote 25, Footnote 26}. The symptoms can include fevers, hallucinations, swollen or rigid limbs, severe inflammation sometimes followed by loss of affected tissues and death^{Footnote 27}. Experimental animal studies indicate the ergot alkaloids act on a number of neurotransmitter receptors which with repeat dosing results in restricted blood flow, particularly of the limbs, weight loss and changes in the levels of some hormones in rats^{Footnote 28}. This study included ergosine, ergocristine and ergocryptine.

4 Fumonisins

Fusarium moniliforme and Fusarium proliferatum are plant pathogens common in grain-growing regions throughout the world. These pathogens can infect grain crops either in the field (pre-harvest) or during storage (post-harvest). The moulds proliferate if grains are grown in hot, dry weather followed by very humid conditions. Mould growth is also favoured by storage under wet conditions. The plant pathogens produce mycotoxins known as fumonisins (FUM). Corn is the grain most vulnerable to fumonisin contamination^{Footnote 29}. The levels of fumonisins can be quite high, even in the absence of visible signs of mould proliferation^{Footnote 30}. There are several forms of fumonisin: fumonisins B1, B2 and B3 are the most prevalent. While studies have focused on fumonisin B1, available data suggests that fumonisins B2 and B3 have a similar toxicological^{Footnote 31, Footnote 32, Footnote 33, Footnote 34}. Fumonisins are heat-stable up to 150°C and are unaffected by mechanical forces (such as grinding), but can be reduced by alkaline treatment (a traditional means of preparing corn masa and other corn-based products such as tacos)^{Footnote 35}.

Although fumonisin contamination is mainly observed in corn, some scientific studies have shown the presence of fumonisins in red wine^{Footnote 36}, sorghum^{Footnote 37}, white beans, wheat^{Footnote 31}, barley^{Footnote 31}, soybeans^{Footnote 31}, figs^{Footnote 31}, rice^{Footnote 38}, black tea^{Footnote 31}, and medicinal herbs^{Footnote 31}.

The ingestion of foods containing fumonisins may be harmful to human health. Health effects which have been observed in specific populations where corn is a major component of the diet and where the climate may favor fumonisin proliferation include esophageal cancer in South Africa and China^{Footnote 30, Footnote 39}, neural tube defects in Central America and the southwestern US^{Footnote 34}. The precise biological effects of fumonisins are complex and relate to their interference with cell metabolism^{Footnote 29}. Experimental animal studies have revealed that fumonisins induce liver and kidney damage in many species^{Footnote 40}. Fumonisin B1 has been classified by IARC as possibly carcinogenic to humans based on evidence in experimental animal studies^{Footnote 41}. This study included fumonisins B1, B2 and B3.

5 Ochratoxin A (OTA)

OTA is a naturally occurring metabolite of Aspergillus and Penicillium moulds. Under favourable moisture and temperature conditions, the fungi can grow on stored material and produce OTA^{Footnote 42}. OTA has been widely detected in cereal grains (wheat, corn, oat, and barley), green coffee, grape juice, beer, wines, cocoa, dried fruits, and nuts^{Footnote 43}. OTA is heat-stable and is only partially destroyed under normal cooking or processing conditions^{Footnote 44}.

The International Agency for Research on Cancer (IARC) has classified OTA as a possible human carcinogen based largely on data from animal studies^{Footnote 45}. The mechanism by which OTA causes kidney tumours in rodents has yet to be fully explained. In animal studies, OTA has also been shown to have effects on the kidneys, the developing fetus, and the immune system. Health Canada completed a risk assessment for OTA, and as a result, has proposed maximum levels for OTA in various food commodities^{Footnote 3} as well as an industry guidance value for OTA in unprocessed cereal grains^{Footnote 3}.

6 Sterigmatocystin (STG)

Sterigmatocystin is a mycotoxin produced mainly by various Aspergillus species. It can also be produced by species such as Bipolaris, Chaetomium, and Emiricella. It has been detected in grains, corn, bread, cheese, spices, coffee beans, soybeans, and pistachio nuts. Wet, warm, conditions favour sterigmatocystin production^{Footnote 46}.

The IARC has classified sterigmatocystin as a possible human carcinogen^{Footnote 47}. It also has properties capable of causing DNA mutations. It is acutely toxic to animals, with the liver and kidneys as its principle targets. This toxin is structurally similar to aflatoxin, however, tests in rats have shown that it is ten times less lethal following acute exposure to high doses and ten to a hundred times less effective at inducing liver cancer^{Footnote 46}. Its human health effects have not been well-studied.

7 Trichothecenes (TRI)

This large family of mycotoxins are typically found in cereal grains (notably wheat, barley, and corn), and have been detected in their derived products (flours, meals, bran, grits, cereals, and beer). These toxins are produced by various species of Fusarium mould in some crops prior to harvest. These toxins are observed in grains suffering from Fusarium head blight (FHB) in the field. Wet, warm weather conditions in the field will favour the development of FHB, and subsequently the production of trichothecenes^{Footnote 48}. The trichothecenes are heat-stable and are only partially destroyed under normal cooking or processing conditions^{Footnote 49}. The most widely commonly occurring trichothenece is DON.

The human health effects of nivalenol^{Footnote 50}, fusarenone^{Footnote 51}, 3-Ac-DON^{Footnote 48}, 15-Ac-DON^{Footnote 48}, neosolaniol (NEO)^{Footnote 48} and DAS^{Footnote 48} are not as well-studied as those of DON. DON is not known to be carcinogenic, but it has been associated with acute and chronic health effects. Outbreaks in Asia, attributed to the consumption of grains with high levels of DON, are associated with short-term human illness, involving nausea, vomiting, abdominal pain, headache and dizziness. In experimental animal studies, long-term exposures to low levels of DON are associated with decreased food intake, weight loss, and effects on the immune system^{Footnote 52}.

T-2 and HT-2 toxins are formed when grain crops remain in the field at or after harvest for extended periods, especially in cold weather, or in grain that becomes wet during storage. They have been detected in wheat, corn, oats, barley, rice, beans, and soybeans and some cereal-based products. Oats are most likely to contain these toxins but they have been detected frequently at lower concentrations in barley. Wheat is only rarely contaminated with these toxins^{Footnote 53}.

The human health effects associated with chronic exposure to HT-2 and T-2 are not known. In animals, these toxins inhibit DNA, RNA and protein synthesis and are cytotoxic. IARC considers HT-2/T-2 toxins not classifiable as to their carcinogenicity to humans based on the lack of available human carcinogenicity data and only limited evidence in experimental animals^{Footnote 54, Footnote 55}. This study examined nivalenol, fusarenone, 3-Ac-DON, 15-Ac-DON, NEO, DAS, DON, HT-2 and T-2.

8 Zearalenone and related compounds

Zearalenone (ZEN) is a mycotoxin produced mainly by Fusarium species. It has been detected in wheat, barley, rice, corn, and other cereals. It is heat-resistant and can be found in finished grain-based products. ZEN is metabolised to α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL)^{Footnote 56, Footnote 57, Footnote 58}.

ZEN is not an acute toxin. ZEN is an estrogenic compound and its major metabolites are more potent estrogenic compounds. It causes infertility in sheep, cattle and pigs, and may lead to earlier sexual maturation in some animals. In experimental animal studies, high oral doses of ZEN have also been shown to be genotoxic, toxic to the liver, and affect blood and the immune system^{Footnote 55, Footnote 59}. IARC concluded that there is limited evidence of the carcinogenicity of ZEN^{Footnote 60}. ZEN has been considered a possible contributing agent in the outbreaks of early puberty in thousands of girls in Puerto Rico and may play a role in human breast and cervical cancer in highly exposed populations^{Footnote 56}.

This study examined ZEN α-ZOL, and β-ZOL.