Aflatoxin M1 Residues in Milk and their Impact on Human Health

Aflatoxins B1 and M1. Biotransformation

Aflatoxin B1 (AFB1) is a toxic secondary metabolite produced by several strains of Aspergillus, basically by Aspergillus flavus and Aspergillus parasiticus. Aspergillus is considered an “storage fungi”: a temperature of 25^OC and a water activity (wa) of 0.83-0.95 are ideal conditions for growth and mycotoxin production.

AFB1 can be found as a natural contaminant in grains and grain by-products (contamination present in DDGS can reach levels 2 or 3 times larger than in the original grain), oilseed cakes (cotton, peanut, coconut, sunflower and others),manioc, silages, feed rations and several human food products such as cereals, dry fruits, sausages, species, wine, legumes, fruits and others.

Aflatoxin M1 (AFM1) is the hydroxilated metabolite (4 hydroxi-aflatoxin B1) of AFB1, and it is the result of metabolism of some animals. In ruminants, part of the AFB1 being consumed through contaminated feed, is transformed in the rumen by the ruminal fluid into aflatoxicol, which is 18 times less toxic than AFB1. This is a reversible process, aflatoxicol can be reverted into AFB1, and it is the reservoir for AFB1 production, thus it is considered dangerous.

The remaining fraction of AFB1 from contaminated feed is absorbed in the gastrointestinal tract and it is hydroxilated into AFM1 in the liver by passive diffusion. AFM1 contaminates milk through blood. Residues in milk can include also non-hydroxilated AFB1.

In dairy cattle complete feedingstuffs, there are two ingredients very susceptible to be contaminated by AFB1: silage and cottonseed. It is very important to test these ingredients and to pay attention to maintain anaerobic conditions during silage production to avoid fungi proliferation since most moulds are aerobic.

In milk producing monogastric animals (sows and others), AFB1 is absorbed in the gastrointestinal tract and taken into the liver where it is metabolized. A fraction of aflatoxin is activated and accumulates in the hepatic tissue. Some water soluble conjugated metabolites of AFB1 are excreted via bile into the feces. Other water soluble metabolites, end-products of AFB1 degradation and non-conjugated metabolites become systemic and can end up contaminating milk. The same process occurs in human milking mothers, putting at risk the health of babies being breastfed.

Toxicity of aflatoxins B1 and M1

Aflatoxins B1 and M1 have a great carcinogenic, teratogenic and mutagenic activities. Their mean effect is on the liver, with affection also of the kidney and brain. They are immunosuppressant: inhibit phagocytosis and protein synthesis by interrupting DNA, RNA and ribosomal proteins production. Amino acid absorption is affected, leading to the increase of amino acid hepatic retention

Aflatoxin M1 residues in milk depending on quantity of aflatoxin B1 ingested

The level of AFM1 residues/day (mg) in milk could reach 2.2% of daily ingestion of AFB1 (mg), with a coefficient of variation between 42 and 59%. Dividing the residue level by milk production (Lts)/cow/day and multiplying by 1000, we obtain the concentration of AFM1 in milk in µg (micrograms)/Lt of milk (ppb). There are other ways of calculating the residue content, but we consider this one the closest to a real situation.

Cows can transform AFB1 into AFM1 within 6 to 24h after consuming complete feedingstuffs contaminated with AFB1. The final concentration of AFM1 in milk depends on breed, initial AFB1 contamination, quantity and duration of feeding, and health status of the animal. The metabolic system of polygastric animals can produce different concentrations of AFM1 in milk with variations by animal and day.

Carcinogenic capacity of Aflatoxins B1 and M1

The carcinogenic potency of AFM1 is significantly less to that of AFB1. They have a TD50 (tumor dose 50) of 10380 ng (nanograms)/kg BW/day and 1150 ng/kg BW/day respectively. So AFM1 is approximately 9 times less carcinogenic than AFB1.

When considering carcinogenic mycotoxins, TDI (tolerable daily intake) can be obtained dividing TD50 by a safety factor, normally between 50 and 50000, with a risk factor of 1/100000. TDI for AFB1 ranges between 0.11 (1150/10000) and 0.19 (1150/6000) ng/kg BW/day. If we divide TD50 for AFM1 by a safety factor or 5000 (10380/5000) we obtain a TDI of 2 ng/kg BW/day, approximately ten times the tolerance for AFB1, which ranges between 0.19 and 0.23.

Babies, children and young are most susceptible to mycotoxins toxicity due to a greater variation in their basal metabolism. They may not have enough biochemical mechanisms for detoxification. In children, brain development continues for years after birth, causing a greater susceptibility to mycotoxins affecting Central Nervous System. For babies and children, milk and dairy products are essential food ingredients and their daily intake is high.

European Union legislation for aflatoxins B1 and M1. Some considerations about dairy products

In the EU, the maximum contamination level allowed for AFB1 in complete and complementary feedingstuffs for dairy animals, is 0.005 mg AFB1/kg of feed (5 ppb) with 12% humidity.

For raw milk, milk for manufacture of dairy products and heat-treated milk for human consumption, maximum AFM1 content is 50 ppt (0.05 µg/kg). In the case of milk for baby formulas and diet products for medical use, the maximum AFM1 content is 25 ppt.

There is no legislation for butter or cheese, but in some EU countries, such Netherlands and Austria, maximum AFM1 content is 200 and 250 ppt for cheese respectively and 20 ppt for butter.

Distribution of AFM1 in different products manufactured with contaminated milk is approximately:

40-60% for cheese, 10% in cream and <2% in butter. The association of AFM1 with casein when it precipitates, provoques that the majority of AFM1 goes to cheese and not whey.

Legislation for aflatoxins M1 in USA, Australia and Mercosur countries (Argentina, Brasil, Paraguay and Uruguay)

In theUSA, Australia, and other countries the maximum AFM1 content for milk, from whole milk to fat-free, is 500 ppt, 10 times the allowed level for EU.

Incidence of contamination with aflatoxin M1 and risk evaluation for human health.

Worldwide studies indicate than average contamination levels for AFM1 in milk of diets from Europe, Latin America, Far East, Middle East and Africa were 0.023, 0.022, 0.360, 0.005 and 0.002 ppb respectively. The values were obtained from 10778, 893, 1191, 231 and 15 samples respectively. In all cases except Far East, values are below EU maximum levels. In the last two cases the number of samples was low and it may not be representative.

When associating the above values to milk consumption in each region (0.29, 0.16, 0.032, 0.12 and 0.042 L/person/day, respectively), AFM1 intake was calculated as 6.7, 3.5, 11.5, 0.6 and 0.085 ng/person/day, respectively. If we consider an average BW of 50kg, TDI would be 0.14, 0.07, 0.23, 0.012 and 0.002 ng AFM1/kg BW/day, respectively. All values would be below the reference value of 2 ng/kg BW/day.

If we consider the average daily consumption in the different World regions mentioned above and we calculate considering a AFM1 contamination with the max. level allowed (0.05 ppb for EU and 0,5 ppb for USA), ingestion of AFM1 would be 15 and 150, 8 and 80, 1.6 and 16, 6 and 60 and 2 and 20 ng/person/day, respectively for the different regions.

If we consider a 50 kg BW person, AFMI intake/kg BW/day would be: 0.3 and 3, 0.16 and 1.6, 0.032 and 0.32, 0.12 and 1.2, 0.04 and 0.4 respectively. All values are below TDI (2 ng/kgBW/day), with exception of the second value for European region.

If we use the same values to calculate for babies of 10 kg BW, the intake would be 1.5 and 15, 0.8 and 8, 0.16 and 1.6, 0.6 and 6, 0.2 and 2 ng/kg BW/day for EU and USA max. levels and for the different World regions respectively. The first value is below TDI (2 ng/kgBW/day), the second value is above TDI previously mentioned, with the exception of the Far East region. The maximum level allowed in USA and other countries for AFM1 is not accepted in the EU.

If we consider a child, 20 kg BW with a daily consumption of 0.5 Lts of milk contaminated with 0.05 ppb or 0.5 ppb of AFM1, the daily intake would be 1.25 or 12.5 ng/kg BW/day respectively. The first value is below TDI for AFM1 in EU, the second value exceeds the max. TDI significantly.

If an average contamination in Europe of 0.023 ppb, resulted in a calculated intake of 6.7 ng/person/day, a contamination of 0.100 ppb (double than max. EU allowed) would result in an intake of 29.13 ng/person/day. For a 50 kg BW person or a 20 kg BW child, the calculated TDI values would be 0.58 and 1.46 ng AFM1/kg BW/day, respectively. Both values are below TDI for AFM1.

Let's look now at the following situation:

Let's apply the equation mentioned in the section “Aflatoxin M1 residues in milk depending on quantity of aflatoxin B1 ingested”. Let's suppose a complete feedingstuff for dairy cattle with a 50% humidity and contaminated with 2.84 µg AFM1/kg (2.84 ppb). Extrapolating to a 12% humidity (reference EU humidity value for mycotoxin contamination), we obtain a contamination of 5 ppb (EU maximum allowed level for AFB1). If we consider a consumption of 30 kg of the mentioned complete feedingstuff (50% humidity)/cow/day and an average milk production of 20 Lts, we obtain an AFB1 intake of 0.0852 mg/cow/day. 2.2% of the intake into the milk result into 0.0019 mg AFM1/cow/day. Dividing by 20 Lts of milk production and multiplying by 1000 we obtain a theoretical milk contamination level of 0.094 µg AFM1/ Lt. That is above the EU maximum allowed level.

The question arises then that using a complete feedingstuff within legal limits we obtain a contamination in milk above legal limits!!. Of course the equation has its limitations, and the values are only approximate, so we should obtain experimental values, but the doubt remains.

Why such large differences between in tolerance levels for AFM1 between EU and other countries?

Even though the general assumption is that AFM1 induces liver cancer in rodents following the same mechanism that AFB1, there are no epidemiological studies establishing a dose-response effect between intake of AFM1, exposure to hepatitis B or C and liver cancer. The additional risk for liver cancer if we consider all milk consumed with a contaminated level of 0.50 ppb for AFM1 (USA and other countries max. level) when compared to 0.05 ppb (EU max. level) is extremely low.

In populations of USA and Western Europe were prevalence of hepatitis B is 1%, the number of additional cases of liver cancer associated with a complete milk contamination of 0.50 ppb versus 0.05 ppb, was of 29 liver cancer cases/1000 million people/year.

Based on these numbers, the controversy remains between the EU and other countries on the max. level allowance for AFM1 contamination in milk.

In 2003, 34 countries favored the 0.05 ppb limit and 22 countries the 0.50 ppb limit.

Could it be that the EU is going too far in establishing such low max. levels of AFM1 contamination?

EU takes extreme precautionary measures because it applies the ALARA principle: As Low As Reasonable Achievable, ie. The max. level should be as low as reasonably possible. The countries allowing for the 0.50ppb level do not follow the same principle.

AFM1 is 10 times less carcinogenic than AFB1, and the mentioned additional risk for liver cancer when max. levels are 0.50 ppb versus 0.05 ppb is very low, but exposure to a genotoxic carcinogenic such as AFM1 is a risk for consumers, in particular for babies, children and youngsters. This fact supports the application of the ALARA principle, which states that for this type of carcinogens, there is no max. dose below which there is no risk for liver cancer. Based on these premises, the EU criteria is correct.

Prevention and Control of aflatoxins M1 and B1

AFM1 is, in general, stable in cheese, yogurt, pasteurized milk, fat-free or whole milk and ice-cream.

It resists some sterilization processes, pasteurization and direct heating processes. In other processes, such as, Roller drying and Spray drying, there can be a reduction of AFM1 between 12 and 86%, depending on the time and temperature exposure for milk. In cheese at 90^OC for 30 minutes, there has been a reduction of 9%. AFM1 can resist lyophilization between -4 and -20^OC for 0,6,12 and 18 months.

Prevention and control of AFB1 will significantly avoid or reduce AFM1 contamination problems in milk and dairy products. It is then very important the application of a HACCP plan for prevention and control of mycotoxins. The problem can be originated though from contaminated raw ingredients.

AFB1 resists temperatures of 120^OC at normal pressure. To try to reduce or eliminate mycotoxins by heat is not effective, practical or economical at industrial level.

Use of fungistatic products (often mistakenly called fungicidal), if effective and of broad spectrum, can help lowering further contamination by AFB1, but it will not affect existing contamination because the product works against the fungi, not the mycotoxin.

An important factor is the use of Anti-Micotoxin Additives, such as phylosilicate clays, esterified glucomannans and others. These additives (mixed with the feed), should be able in the gastrointestinal tract to form irreversible complexes with beta-cetolactone and alfa-bislactone groups of the AFB1 molecule. These non toxic complexes will be then eliminated in the feces.

Analytical methods for aflatoxin M1 in milk and dairy products

The most recommended method is the use of immunoaffinity columns with monoclonal antibodies specific for AFM1, followed by detection and quantification by HPLC.

Regarding the ELISA method, it has been validated by the Community Bureau of Reference after a series of comparison studies with the HPLC method. It can even be used in the farm.

With these methods, the limits for detection and quantification range between 0.005 and 0.01 ppb, respectively.

Comments

We should keep the risk level as low as possible and be always alert, but not alarmed. The situation for AFM1 is, at least in Europe, under control and with a low risk level. Evidently there can be some exceptions.

Considering public health risk, EU maintains the max. level of 0.05 ppb for AFM1 in milk and 0.025 ppb for dairy products for lactating babies.

Some of the arguments between the EU Scientific Committee and Scientific Committees of other countries that defend the higher max. level of 0.5 ppb for AFM1 in milk were as follows:

EU Sc. Committee indicated that risks associated to exposure to this mycotoxin should be carefully considered because of the high intake of milk and dairy products by babies and children.

The countries defending a 0.5 ppb max. level, argued that there could be important economic losses due to the difficulty to export milk to countries following the lower max. level. But no study with detailed numbers on the economic consequences was presented.

Some countries delegations claimed that the 0.05 ppb level was difficult to achieve in several world regions and that a 0.5 ppb level was enough to guarantee public health, because that level could be reasonably achieved worldwide.

In addition, it was stated that in developing countries, there could be a shortage of milk with negative consequences in nutrition if a level lower than 0.5 ppb was established. There were even some delegations claiming that 0.5 ppb for AFM1 should be considered the minimum level achievable due to management of max. concentrations of AFM1 in feedstuffs.

The EU Scientific Committee for Human Health answered that this claim did not follow the recommendations mentioned in the Code of Good Practices for Reduction of AFB1 in raw ingredients, complete and complementary feedingstuffs for milk producing livestock and adopted by the Commission of the Codex Alimentarius, from which we are literally reproducing some extracts:

“If aflatoxin B1 is detected, consider one or more of the following options.

1. In all cases ensure that the aflatoxin B1 level of the finished feed is appropriate for its intended use (i.e. maturity and species of animal being fed) and is consistent with national codes and guidelines or qualified veterinary advice.

2. Consider the restriction of AFB1 contaminated feed to a percentage of the daily ration such that the daily amount of AFB1 ingested would not result in significant residues of AFM1 in milk.

3. If feed restriction is not practical, divert the use of highly contaminated feedingstuffs to non-lactating animals only”

Let's remember that EU legislation establishes a max. level of 5 ppb AFB1 in complete and complementary feedingstuffs (with a humidity of 12%) for dairy livestock. But dairy livestock do not consume only complementary feedingstuffs, dairy cattle for example normally consume 23 – 27% complementary feedingstuff , 45 – 50% silage and the rest of the diet is composed of cottonseed, brewery by-products, hay, etc. Thus, contamination by AFB1 in the complete feedingstuff could come from complementary feedingstuff and/or the rest of the ingredients. If the final ration is labeled with the term “complete feedingstuff”, then we could establish that the max. level of 5 ppb of AFB1 (with a 12% humidity) should apply to the complete feedingstuffs being prepared by the farmer.

Considering that the complete feedingstuffs for dairy cattle have a higher humidity than 12%, we should take into account this factor and analyze not only AFB1 but also humidity and to extrapolate the result to the regulated 12% humidity. In this way we can compare our values with the EU legal limits.

References

1.- Gimeno, A. (2011). El impacto negativo de algunas micotoxinas en el ganado vacuno lechero

(http://www.engormix.com/MA-ganaderia-leche/sanidad/articulos/micotoxinas-en-el-ganado-vacuno-lechero-t3029/165-p0.htm). En www.engormix.com (Sección Micotoxinas en Español).

2.- Gimeno, A. (2005). Aflatoxina M1 en la Leche. Riesgos para la Salud Pública. Prevención y Control

(http://www.engormix.com/MA-micotoxinas/articulos/aflatoxina-leche-riesgos-salud-t372/p0.htm)

En www.engormix.com (Sección Micotoxinas en Español).

3.- Gimeno, A. (2002). Residuos de Aflatoxina M1 y otras micotoxinas en leche y derivados; control y recomendaciones (http://www.engormix.com/MA-ganaderia-leche/sanidad/articulos/residuos-aflatoxina-otras-micotoxinas-t114/165-p0.htm). En www.engormix.com (Sección Micotoxinas en Español).

4.- Gimeno, A.; Martins, M.L. (2011). “Micotoxinas y Micotoxicosis en Animales y Humanos”. 3ª Edición. Special Nutrients, Inc. USA (Ed.). pp. 1-128. The author can be asked for the manual (gimenoalberto@hotmail.com).

5.- Gimeno, A; Martins, M.L. (2012). “Mycotoxins and Mycotoxicosis in Animals and Humans”. 2nd Edition. Special Nutrients, Inc. USA (Ed). pp. 1-149. the author can be asked for the manual. (gimenoalberto@hotmail.com).

6.- Raquel Rubio Martínez (2011) Incidencia de aflatoxinas en leche de oveja y derivados en Castilla-La Mancha. Tesis Doctoral. pp.1-246. Download the pdf file from www.google.com searching for “Tesis Doctoral de Raquel Rubio Martinez”

Note:

The present article was base for the conference about this subject in the III Congreso de Alimentación Animal: Seguridad Alimentaria y Producción de Alimentos, held on December 3rd and 4th in Bilbao (Euskadi), Spain. (www.congresoalimentacionanimal.com).

In agreement with the Author and the Congress Directive Committee, the article is published in Engormix (www.engormix.com) (Mycotoxins in English).

Annex to the article “Aflatoxin M1 residues in milk and their impact on human health.”

Annex to article published in the proceedings of the III Congreso de Alimentación Animal: Seguridad Alimentaria y Producción de Alimentos, December 3rd and 4th, 2013, Bilbao (Euskadi), Spain.

The spanish version of the article is also published in www.engormix,com (Engormix Spanish/ Mycotoxins Community)

According to the equation mentioned in the article referring to the approximate aflatoxin M1 (AFM1) contamination in milk based on the original aflatoxin B1 (AFB1) intake (Patterson et al,1980; Van Egmond, 1989):

The level of AFM1/day (mg) in milk could reach 2.2% of the daily intake of AFB1 (mg), with a coefficient of variation between 42 and 59%. Dividing the calculated result by milk production/cow/day and multiplying by 1000, we obtain an approximate AFM1 concentration in µg/Lts of milk (ppb).

Let's write the equation in the following manner and adjust all calculations to dry matter.

C= (AFB1 x RF x 0,022)

Were:

C= AFM1 concentration in milk (micrograms/Liter/kg).

AFB1= AFB1 concentration (micrograms/kg) found in the complete feedingstuff (based on dry matter).

RF= complete feedingstuffs intake (kg/cow/day) (based on dry matter).

L = liters of milk produced/cow/day.

EU legislation establishes in complete and complementary feedingstuffs for dairy animals a max. AFB1 contamination of 5 µg (micrograms)/kg (ppb) with a 12% humidity of the substrate, which represents 5.68 ppb over dry matter.

Table 1 shows different calculated results of AFM1 contamination in milk, based on AFB1 intake with a theoretical complete feedingstuff contaminated with 4.8 ppb over dry matter (within EU limits).

TABLE 1,- Calculated AFM1 contamination in milk based on AFB1 intake and milk production.

Aflatoxin M1 Residues in Milk and their Impact on Human Health - Image 1

Click here to enlarge the image

AFB1 = aflatoxin B1 concentration (microgramos/kg) in complete feedingstuff (over dry matter).

EU AFB1 = European Union max. AFB1 level (over dry matter).

L = liters of milk produced/cow/day.

RF= complete feedingstuff intake (kg/cow/day) (over dry matter).

C= calculated AFM1 concentration in milk (micrograms/Liter/kg).

ppb = micrograms/Liter/kg, or µg/Liter/kg.

We can clearly observe that, even though the AFB1 contamination of the complete feedingstuffs is within EU limits, the milk produced is susceptible to be contaminated with AFM1 above max. EU levels (0,05 ppb or 0.025ppb for baby formulas). These calculations point out the argument exposed in the section “Incidence of contamination with aflatoxin M1 and risk evaluation for human health.” of the original article.

As mentioned in the article the equation has its limitations, and the values are only approximate, so we should obtain experimental values, but the doubt remains.

The parameters “liters of milk/cow/day” and “complete feedingstuff intake kg/cow/day (over dry matter)”, were obtained from published studies where the values for such parameters could be obtained from the nutritional characteristics of the complete feedingstuffs.

References

Van Egmond, H.P. (1989). Aflatoxin M1: Ocurrence, Toxicity, Regulation, in Mycotoxins in Dairy Products. Hans P.Van Egmond (Ed.) Elsevier Applied Science, London and New York. Chapter 2, pp.11-55.

Patterson, D.S.; Glancy, E.M.; Roberts, B.A. (1980). The carry-over of aflatoxin M1 into the milk of cows fed rations containing a low concentration of aflatoxin B1. Food Cosmet. Toxicol. 18: 35-37.

Aflatoxin M1 Residues in Milk and their Impact on Human Health

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