Risk assessment of Aflatoxin B1 from the consumption of different foods in South East Asia region (A case study based on Bangladesh food consumption data)

- Dr. Kshitij Shrestha, SFRO, DFTQC

1. Hazard Identification

Aflatoxins are bisfuranocoumarin compounds produced primarily by toxigenic strains of the fungi Aspergillus flavus and Aspergillus parasiticus. A. parasiticus produces AFB1, AFB2, AFG1 and AFG2, whereas A. flavus mainly produces AFB1 and AFB2 (EFSA, 2007).

When concentrations or maximum limits mention ‘total’, it typically refers to the sum of AFB1, AFB2, AFG1 and AFG2. The most frequently found Aflatoxin in contaminated food samples is AFB1 and the three others are generally not reported in the absence of AFB1 (FAO/WHO, 2018).

The Aflatoxin-producing fungi are found especially in areas with a hot, humid climate and Aflatoxins are found in food as a result of both pre- and post-harvest fungal contamination. The rate and degree of contamination depends on temperature, humidity, soil and storage conditions (EFSA, 2007).

For this case study, the risk assessment of Aflatoxin B1 from the consumption of different foods has been carried out based on the Bangladesh food consumption data.

2. Hazard characterization

The available toxicological knowledge on Aflatoxins is mostly related to AFB1. Aflatoxins are genotoxic and the critical effect of Aflatoxins in all the previous assessments was liver cancer. Following absorption, Aflatoxins undergo first pass metabolism in the liver where they exert their toxicity due to the formation of toxic metabolites. In addition, AFB1 induces point mutations, mitotic recombination in mammalian cells and genetic instability (IARC, 2012). 

Co-exposure to hepatitis viruses, in particular hepatitis B, has a strong influence on the carcinogenic risk of Aflatoxins to humans. In epidemiological studies, there is an interaction between Aflatoxin exposure and hepatitis B infection, and subjects positive for hepatitis B surface antigen (HBsAg) show a multiplicative risk for liver cancer when present together with Aflatoxin exposure (FAO/WHO, 2018). IARC (2012) classified Aflatoxins as a group of carcinogenic to humans (Group 1) causing hepatocellular carcinomas (HCCs).

The double bond in the furan ring of AFB1 and AFG1 can be oxidised and forms an 8,9-exo-epoxide that readily reacts with DNA and other nucleophiles (FAO/WHO, 2018). AFB1 forms DNA adducts by covalent binding to N7-guanine, resulting in persistent DNA lesions. These lesions may subsequently lead to transversion mutations (IARC, 2012).

3. Exposure Assessment

3.1 Occurrence data

The food contamination data for Aflatoxin B1 for all the regions in the world was downloaded from World Health Organization (WHO) Food Safety Collaborative Platform (FOSCOLLAB) database and the summary statistics for all the region is given in the Appendix A1. The total of 1,28,471 number of contamination data were obtained for different food groups. The summary statistics for Aflatoxin B1 contamination data from the South East Asia region is given in the Appendix A2.

3.2 Data analysis

The left-censored data (results below the LOD or below the LOQ) were treated by the substitution method as recommended in ‘Principles and Methods for the Risk Assessment of Chemicals in Food’ (WHO/IPCS, 2009). The same method is indicated in the EFSA scientific report ‘Management of left-censored data in dietary exposure assessment of chemical substances’ (EFSA, 2010) as an option for the treatment of left-censored data. The guidance suggests that the lower bound (LB) and upper bound (UB) approach should be used for chemicals likely to be present in the food (e.g. naturally occurring contaminants, nutrients and mycotoxins). The LB is obtained by assigning a value of zero (minimum possible value) to all samples reported as lower than the LOD (< LOD) or LOQ (< LOQ). The UB is obtained by assigning the numerical value of LOD to values reported as < LOD and LOQ to values reported as < LOQ (maximum possible value), depending on whether the LOD or LOQ is reported by the laboratory. 

3.3 Food consumption data

The food consumption data was obtained from Global Individual Food Consumption Data (GIFT) from Food and Agriculture Organization (FAO). Among the South East Asian countries, The National food consumption nutrition survey latest data is available for Bangladesh (2017-18). The data contains comprehensive national survey on food consumption and is a good representation of food consumption pattern with rice as a staple food. The food consumption pattern of Bangladesh is similar to India and Nepal, and hence the latest food consumption data of Bangladesh was chosen for this study.

 The data includes the food consumption data from 3457 consumers. To carry out the exposure assessment for different age groups, the consumer was classified based on the age category (infants: < 12 months old, toddlers: ≥ 12 months to < 36 months old, other children: ≥ 36 months to < 10 years old, adolescents: ≥ 10 years to < 18 years old, adults: ≥ 18 years to < 65 years old, elderly: ≥ 65 years to < 75 years old, very elderly: ≥ 75 years old) as per guidance from EFSA Panel on Contaminants in the Food Chain (CONTAM) (EFSA, 2020).

Out of total 3457 consumers, the number of infants, toddlers, other children, adolescents, adults, elderly and very elderly were 60, 263, 620, 399, 2021, 68 and 26 respectively. The bodyweight data for 80 subjects were not available. The regression equation from the rest of the subject up to the age of 50 years was used to estimate the body weight of those 80 subjects for the calculation.

The summary statistics of the food consumption data for infants (Appendix B1), toddlers (Appendix B2), other children (Appendix B3), adolescents (Appendix B4), adults (Appendix B5), elderly (Appendix B6) and very elderly (Appendix B7) are given in the Appendix B1, Appendix B2, Appendix B3, Appendix B4, Appendix B5, Appendix B6 and Appendix B7 respectively.

3.4 Food classification

Consumption and contamination data were classified according to the FoodEx2 classification system (EFSA, 2011). FoodEx2 is a food classification system that was developed by EFSA in 2009 with the objective of simplifying the linkage between occurrence and food consumption data when assessing the exposure to hazardous substances. The system consists of a large number of individual food items aggregated into food groups and broader food categories in a hierarchical parent–child relationship.

3.5 Exposure assessment

To calculate chronic dietary exposure to Aflatoxins, food consumption and body weight data at the individual level were accessed in the comprehensive database. Occurrence data and consumption data were linked at the relevant FoodEx2 level. In addition, the different food commodities were grouped within each food category to better explain their contribution to the total dietary exposure to Aflatoxins.

The exposure assessment was carried out for different scenarios:

1. Exposure assessment for different age groups
2. Exposure assessment based on contamination data from all the regions
3. Exposure assessment based on the contamination data having less than 2ppb Aflatoxin B1 contamination level (as per the European legislation) (The contamination data with more than 2ppb level contamination was filtered out)
4. Exposure assessment based on the contamination data only from the South East Asia region
5. The contamination data showed large portion of left censored data (not detected), and hence it is likely that the exposure assessment could be either under or over estimated. To accommodate this fact, all the exposure assessment calculations were done for both the lower bound and upper bound scenarios. For the lower bound scenarios, the not detected left censored data were assumed to have zero Aflatoxin B1 concentration while, for the upper bound scenarios, the not detected left censored data were replaced by 0.1 ppb (as LOD/LOR for not detected values in the contamination data).

The consumption data was filtered for different age group and the exposure assessment was carried out separately for the different age groups.

At first, the consumption data was aggregated for same survey day, same subject (individual) and same FoodEx2 code at level 4 to calculate gram per person per day consumption. The data was filtered for the age group and was linked with body weight of the respective subject. The data was spread for different FoodEx2 group making each of them in a new column. Bootstrapping method was used for the Monte Carlo simulation. For that, row wise random sampling (bootstrapping) from the dataset was carried out to create the data with rows equivalent to the number of iterations 1,00,001. The consumption data for different food groups were converted into grm per kg body weight per day by dividing with the subject (individual) body weight.

Out of 1,28,471 Aflatoxin B1 contamination dataset, 35,912 data were reported with FoodEx2 level 3 or below and hence, the contamination data was not specific to the food products. Those data were removed from the contamination data source. The total of 92,559 contamination data for Aflatoxin B1 was selected from all the regions having FoodEx2 information at least up to level4. The summary statistics of the contamination data is given in Appendix A1. The contamination data was filtered as per the different scenarios described above. Bootstrapping or random sampling method was used to have 100001 contamination data for each FoodEx2 level 4 and was spread to obtain data in different column for each FoodEx2 group.

After having 1,00,001 bootstrapped data for consumption and contamination for each FoodEx2 category, the exposure assessment was calculated by multiplying the consumption data with contamination data for the matching FoodEx2 code (same food group) for each iteration. The obtained exposure value was aggregated for same subject (same iteration) to calculate the exposure at FoodEx2 level3, level 2, level1 food category and the total exposure from all food. All these calculations were automated by coding in R script. The exposure assessment calculation was carried out in R software (version 4.4.2).

4 Risk characterization

The general principles of the risk assessment process for chemicals in food was applied as described by WHO/IPCS (2009), which include hazard identification and characterization, exposure assessment and risk characterization. In addition, EFSA guidance pertaining to risk assessment of substances which are both genotoxic and carcinogenic has been applied for the present assessment (EFSA,2005).

In view of the genotoxic properties of Aflatoxins, it was not appropriate to establish a tolerable daily intake and considered the possibility of applying a Margin of Exposure (MOE) approach. Based on studies in animals, the BMDL10 of 0.4 μg/kg bw per day for the incidence of hepato cellular carcinoma (HCC) was used in a margin of exposure (MOE) approach for the risk characterization, as suggested by EFSA CONTAM Panel. An MOE of 10,000 and above, generally indicates a low level of concern for a substance, especially when it comes to genotoxic and carcinogenic effects (EFSA, 2020).

The calculation of a BMDL from the human data was not appropriate and no MOE approach could be used for these data; instead, the cancer potency estimates reported by JECFA were used.

4.1 Risk characterization based on animal data (dietary exposure assessment and margin of exposure)

The dietary exposure of Aflatoxin B1 and MOE for FoodEx2 level 1 based on the contamination data from all the region for different age group is shown in Appendix C1. This table provide the exposure from ten different food groups:

1. Grains and grain-based products
2. Vegetables and vegetable products
3. Starchy roots or tubers and products thereof, sugar plants
4. Legumes, nuts, oilseeds and spices
5. Fruit and fruit products
6. Meat and meat products
7. Fish, seafood, amphibians, reptiles and invertebrates
8. Milk and dairy products
9. Eggs and egg products
10. Animal and vegetable fats and oils and primary derivatives thereof

For the genotoxic and carcinogenic compounds, the MOE below 10,000 value is considered to have public health concern. At 90 percentile exposure (P90) under lower bound scenarios, the seven food categories (Starchy roots or tubers and products thereof, sugar plants, Fruit and fruit products; Meat and meat products; Fish, seafood, amphibians, reptiles and invertebrates; Milk and dairy products; Eggs and egg products; and Animal and vegetable fats and oils and primary derivatives thereof) contributed very low exposure of Aflatoxin B1 with MOE above 10,000 indicating low public health concerns. However, in the upper bound scenarios, exposure is calculated to be significant enough to have MOE below 10,000.

At 90 percentile exposure (P90) under lower bound scenarios, the three food categories (Grains and grain-based products; Vegetables and vegetable products; and Legumes, nuts, oilseeds and spices) contributed significant exposure of Aflatoxin B1 with MOE far below the 10,000 indicating high public health concerns. The low MOE value is mainly observed for the grains and grain-based products for all the different age groups (infants, toddlers, other children, adolescent, adults, elderly and very elderly). The highest exposure was found for mainly the other children and adolescent group of population. For the infants, milk being the major food source, is known to contribute in the Aflatoxin exposure in the form of Aflatoxin M1. That part is out of the scope of this study.

With the observation of significant exposure of Aflatoxin B1 for all the age group from mainly the three categories of food, it was necessary to further investigate which food is mainly contributing the exposure within that food category. Hence, the exposure assessment was compared at fourth level of FoodEx2 for different age group for different scenarios and the results have been presented in Appendix C2. Foods which contributed low exposure (having MOE above 10,000) have not been shown in the table. For the contamination data, three results from three scenarios have been shown:

1. Exposure assessment based on the contamination data from all around the world
2. Exposure assessment from the contamination data with only lower than 2ppb contamination level
3. Exposure assessment based on the contamination data from only the South East Asia

For all the age group, rice was found to be the dominant source of Aflatoxin B1 exposure, mainly due to high rice consumption pattern in the population.

For infants, grains and grain based products contributed around 2.1 to 3.5 ng/kg bw/day exposure at 95 percentiles and all of them were found to be contributed by rice. This corresponds to the MOE value in the range of 114 to 192, which could be a serious public health concern. The exposure was similar even when contamination data from South East Asia was only considered. Even if the contamination level of Aflatoxin B1 would be reduced to below 2ppb, the exposure at 95 percentiles is still likely to be at the range of 1.3 to 2.2 ng/kg bw/ day.

For toddlers, grains and grain based products contributed 9.2 ng/kgbw/day exposure at 95 percentiles and all of them were found to be contributed by rice. This corresponds to the MOE of 44 which could also be a serious public health concern. The exposure was similar even when contamination data from South East Asia was only considered. When the contamination data above 2ppb were filtered, the exposure was reduced to around 5 ng/kg bw/ day with MOE of around 80.

For other children, grains and grain based products contributed 14.9 ng/kgbw/day exposure at 95 percentiles and all of them were found to be contributed by rice. This corresponds to the MOE of 27 which could also be a serious public health concern. The exposure was similar even when contamination data from South East Asia was only considered. When the contamination data above 2ppb were filtered, the exposure was reduced to around 8.2 ng/kg bw/ day with MOE of around 49.

For adolescent, grains and grain based products contributed 11.2 ng/kgbw/day exposure at 95 percentiles and all of them were found to be contributed by rice. This corresponds to the MOE of 36 which could also be a serious public health concern. The exposure was similar even when contamination data from South East Asia was only considered. When the contamination data above 2ppb were filtered, the exposure was reduced to around 6.3 ng/kg bw/ day with MOE of around 64.

For adults, grains and grain based products contributed 9.9 ng/kgbw/day exposure at 95 percentiles and all of them were found to be contributed by rice. This corresponds to the MOE of 40 which could also be a serious public health concern. The exposure was similar even when contamination data from South East Asia was only considered. When the contamination data above 2ppb were filtered, the exposure was reduced to around 5.4 ng/kg bw/ day with MOE of around 74.

For elderly, grains and grain based products contributed 9.4 ng/kgbw/day exposure at 95 percentiles and all of them were found to be contributed by rice. This corresponds to the MOE of 43 which could also be a serious public health concern. The exposure was similar even when contamination data from South East Asia was only considered. When the contamination data above 2ppb were filtered, the exposure was reduced to around 5.2 ng/kg bw/ day with MOE of around 77.

For very elderly, grains and grain based products contributed 8.4 ng/kgbw/day exposure at 95 percentiles and all of them were found to be contributed by rice. This corresponds to the MOE of 48 which could also be a serious public health concern. The exposure was similar even when contamination data from South East Asia was only considered. When the contamination data above 2ppb were filtered, the exposure was reduced to around 4.7 ng/kg bw/ day with MOE of around 86.

These observations raises a serious concern on the possibility of rice being the major contributor to the Aflatoxin exposure in the South East Asian populations such as Nepal, India, Pakisthan, Bangladesh, Thailand, Vietnam, Srilanka etc, where rice is the staple diet.

A general misconception among the technologist persist that Aflatoxin is mainly the concern of maize and peanuts and rice is safe to eat. This misconception has shifted the focus of carrying out the surveillance of Aflatoxin in other food products except rice. In the Nepalese context, the mandatory standards allow up to 20 ppb of mycotoxin legally. However, we have already observed above that even the exposure from the contamination data below 2 ppb of Aflatoxin B1 could have significant public health concern due to its genotoxic and carcinogenic potential. This is a clear underestimation of the potential cancer risk from the food consumption by the food competent authorities of the South East Asian countries.

4.2 Risk characterisation based on human data (Cancer risk)

The EFSA CONTAM Panel used the cancer potency estimates reported by JECFA for the risk characterization. Using model averaging, JECFA calculated potency estimates of 0.017 (mean) and 0.049 (95% UB) per 100,000 person-years per ng/kg bw per day for HBsAg-negative individuals and 0.269 (mean) and 0.562 (95% UB) per 100,000 person-years per ng/kg bw per day for HBsAg-positive individuals. Considering the new evidence regarding HCV as a risk factor, the EFSA CONTAM Panel decided to take also the prevalence of HCV into account in the risk characterization.

As described in FAO/WHO (2018), the Aflatoxin-related hepatocellular carcinoma risk is estimated from the cancer potency estimates using the following equation:

R= (PHBV +) x (AFexposure) x (HBV +) + (PHBV -) x (AFexposure) x (1 - HBV +)

where R is the cancer risk

PHBV+ is the potency estimates P for the HBV+ fraction of the population

PHBV− is the potency estimates P for the HBV− fraction of the population

HBV+ is the population fraction of chronic HBV cases

Using the conservative approach, the prevalence of HBV/HCV in Bangladesh was estimated to be in the range of 3.2 to 6% (Banik,2022). The estimated cancer risk per 10000 populations for different age group based on the exposure due to the contamination data of all the region using mean and 95% UB cancer potency data for mean and P95 exposure data is shown in Appendix C3.

To put the cancer risk estimates into context, the WHO Guideline for drinking-water quality (WHO, 2011) was used. According to this guideline, an excess lifetime cancer risk of 10E−5 or less is considered to be of low risk for health concern. Assuming a lifetime expectancy of 70 years, this corresponds to a yearly excess cancer risk of 0.014 additional cancer cases per 100,000 subjects. Comparing the estimated AFB1-induced cancers calculated with this yearly excess cancer risk, ten to hundreds of times higher risk is identified when using the mean dietary exposure and the P95 dietary exposure. This observation clearly shows the significant cancer risk health concerns for the South East Asian population from the exposure of Aflatoxins mainly from the rice source.

It’s time to rethink, are we doing enough to protect the consumer from the cancer risk due to Aflatoxins exposure from the food, mainly the rice?

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