Health risk assessment of aflatoxin M1 in infant formula milk in IR Iran

Highlights

• Developing two accurate, precise, selective and sensitive methods for determining aflatoxin M1 (AFM1) in infant formula milk.

• Application of the methods on real infant formula milk samples.

• Additional cancer risk due to mean and median exposure to AFM1 in infant <6 months were 0.00010 and 0.00012 additional cases per year per 10⁵ individuals, respectively.

• HI values for the mean and median exposure to AFM1 for infants were quite below one which indicates no health concern.

• Necessity of cumulative risk assessment of AFM1in infants from different provinces in IR Iran consuming infant formula milk along with milks from other sources.

Abstract

In this study, two accurate, precise, selective and sensitive methods were developed for determining aflatoxin M1 (AFM1) in infant formula milk using immunoaffinity column clean-up followed by high performance liquid chromatography (HPLC) with fluorescence detection. The validated methods were used for determination of AFM1 in 29 samples of 6 different infant formula milk brands and the risk of AFM1 in infants aged zero to 6 months old was assessed using cancer risk, Margin of Exposure (MOE) and Hazard Index (HI). Only one sample (3.4%) was contaminated with AFM1. Although the results showed that MOE values for the mean and median exposure to AFM1 was <10,000 in infants, the additional cancer risk due to mean and median exposure to AFM1 in infant <6 months were 0.00010 and 0.00012 additional cases per year per 10⁵ individuals, respectively, which indicates no health concern. In addition, HI values for the mean and median exposure to AFM1 for infants were quite below one which indicates no health concern. To the best of our knowledge, this is the first report on risk assessment of AFM1 in infant formula milk consumed by Iranian infants <6 months old, presenting a low risk for the evaluated groups.

Introduction

The aflatoxins are a group of mycotoxins produced by certain Aspergillus species, in particular, Aspergillus flavus and Aspergillus parasiticus that contaminate plants and their products (Yazdanpanah and Eslamizad, 2015; Yazdanpanah, 2006). Four major aflatoxins including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1, and aflatoxin G2, and two metabolic products including aflatoxin M1 (AFM1) and aflatoxin M2 are recognized as direct contaminants of foods and feeds. Aflatoxins are known hepatotoxic, mutagenic, carcinogenic, teratogenic, and immunosuppressive toxins (EFSA 2005; Herzallah, 2009). AFB1 is the most potent hepatocarcinogen, which is classified by International Agency for Research on Cancer (IARC) as a Group 1 carcinogen (IARC, 2002; Cavaliere et al., 2005). Aflatoxin M1 (AFM1) is the monohydroxylated derivative of AFB1 that is excreted in the human and animal milk following consumption of AFB1 contaminated food. AFM1 is relatively stable either to thermal processes like sterilization and pasteurization or during preparation and storage of different dairy products (Cavaliere et al., 2005). Even though the toxicity of AFM1 is less than AFB1, it has been classified as probable human carcinogens Group 2B by IARC (IARC, 2002).

AFM1 is found in milk and dairy products including yogurt, cheese, milk powder, ice cream, and regular cream. In IR Iran, there are a lot of information about contamination of milk with AFM1 (Ghaffarian Bahraman et al., 2019; Abyaneh et al., 2019; Fooladi Moghaddam et al., 2019; Khaneghahi Abyaneh et al., 2020; Rahmani et al., 2018; Khaneghahi Abyaneh et al., 2019; Kamkar et al., 2014; Yazdanpanah, 2006). In addition, infant formula milk which is one of the important products manufactured from milk is contaminated with AFM1 (Quevedo-Garza et al., 2020; Meucci et al., 2010; Kim et al., 2000; Srivastava et al., 2001).

High milk consumption by all age groups and especially by children seems to be one of the most important sources of dietary exposure for AFM1 (Tsakiris et al., 2013). After birth, human milk is the first choice for the newborn, but the role of infant formula milks is considerable, when breastfeeding is not possible, not recommended or not sufficient (Meucci et al., 2010). Therefore, many countries have established maximum limits for AFM1 in infant formula. For example, the maximum limits for AFM1 in infant formula in the European Union and USA are 25 pg/g and 50 pg/g, respectively (Commission Regulation, 2006; FDA, 2005; Jawaid et al., 2015). In IR Iran, the National Standard Organization (NSO) has established the maximum limit of AFM1 at 25 pg/g for infant formula milks (ISIRI, 2002). There are several HPLC methods were published for the determination of AFM1 in infant formula (Quevedo-Garza et al., 2020; Ivastava et al., 2001; Kim et al., 2000; Meucci et al., 2010). Considering the probable human carcinogenic effect of AFM1 on one hand and high infant formula consumption by infants on the other hand, determination of this contaminant in infant formula using sensitive, accurate, precise, and selective method is necessary.

In this study, two selective, sensitive, accurate, and precise HPLC methods for determining of AFM1 in infant formula milk were developed and validated, and performance characteristics of the methods compared in terms of matrix effect, accuracy, precision, and limits of detection and quantification. In these methods, HPLC monolithic column with a fluorescence detector, and immunoaffinity column (IAC) as a clean-up procedure were used and matrix effect was investigated in infant formula samples. To establish the capability and the suitability of these methods, a variety of infant formula milk samples were analyzed for the presence of AFM1, and risk of AFM1 was assessed for infants 0–6 months old by calculation of Hazard Index (HI), Margin of Exposure (MOE) and cancer risk considering the prevalence of Hepatitis B Virus (HBV).