Risk assessment of lead and cadmium concentrations in hen's eggs using Monte Carlo simulations

Abstract The hen egg is one of the main sources of cheap, great quality, and nutritious food. This study aimed at determining the level of lead (Pb) and cadmium (Cd) in hen eggs and at assessing the carcinogenic and non‐carcinogenic risks caused by the consumption of hen eggs collected in Iran. A total of 42 hen eggs from 17 major brands were randomly sampled from supermarkets. Lead and cadmium concentrations were determined by using inductively coupled plasma mass spectrometry (ICP‐MS). Additionally, using the Monte Carlo simulation (MCS) method to calculate dietary exposure, target hazard quotient (THQ), and incremental lifetime cancer risk (ILCR), the related human health risk associated with ingesting these hazardous metals for adults was evaluated. The average Pb and Cd concentrations in whole eggs were 7.16 ± 0.248, and 2.83 ± 0.151 μg kg−1, respectively, which were less than the maximum permitted levels, established by FAO/WHO and the Institute of Standards and Industrial Research of Iran (ISIRI). Pb and Cd concentrations were significantly correlated at the 0.05 level (r = 0.350). Regarding the levels of Pb and Cd in eggs, overall, the estimated weekly intake (EWI) of these metals for adults by egg consumption was determined 0.014 and 0.007 mg/week, respectively, which were lower than the risk values suggested. The carcinogenic and non‐carcinogenic indexes of Cd and Pb indicated that the adult population in Iran was safe (THQ Pb and Cd <1, ILCR <10−6 Pb). It should be emphasized that this research primarily focuses on egg consumption, which may account for a relatively small portion of Iranian consumers' overall exposure to Pb and Cd. Therefore, a comprehensive study on the risk assessment of these metals through whole‐diet foods is recommended. The findings showed that lead and cadmium levels in all evaluated eggs were suitable for human consumption. Adults' Pb and Cd exposure from eating eggs was significantly lower than the risk levels established by Joint FAO/WHO Expert Committee on Food Additives (JECFA), per the exposure assessment. According to the THQ values of these dangerous metals, which were below one value, egg eating by Iranian consumers does not present a non‐carcinogenic risk. In addition, this finding provides accurate and reliable information for policymakers to improve food safety status to reduce public health hazards.

The carcinogenic and non-carcinogenic indexes of Cd and Pb indicated that the adult population in Iran was safe (THQ Pb and Cd <1, ILCR <10 −6 Pb). It should be emphasized that this research primarily focuses on egg consumption, which may account for a relatively small portion of Iranian consumers' overall exposure to Pb and Cd.
Therefore, a comprehensive study on the risk assessment of these metals through whole-diet foods is recommended. The findings showed that lead and cadmium levels in all evaluated eggs were suitable for human consumption. Adults' Pb and Cd exposure from eating eggs was significantly lower than the risk levels established by Joint FAO/WHO Expert Committee on Food Additives (JECFA), per the exposure assessment. According to the THQ values of these dangerous metals, which were below one value, egg eating by Iranian consumers does not present a non-carcinogenic risk. In

| INTRODUC TI ON
One of the primary sources of affordable, high-quality, and nutrientdense protein with a variety of mineral salts and vitamins is the hen egg (Talakesh et al., 2020). The World Health Organization (WHO) states that hen eggs are an essential part of the human diet, but that their consumption is higher than that of other sources of animal proteins because of economic issues Talakesh et al., 2020).
The Food and Agriculture Organization (FAO) predicts that egg consumption would rise from 6.5 to 8.9 kg per year in impoverished countries to 13.5 to 13.8 kg per year in industrialized countries (Talakesh et al., 2020). The World Health Organization (WHO) recommended that for a requirement of 2100 kcal consume 60 g of eggs on average twice or 4 times a week (Giannetto et al., 2016) The average annual per capita consumption of hen eggs in the world is 250 Kg. However, per capita, egg consumption among households in Tehran is much lower than the recommended standards (Talakesh et al., 2020). Iranians consume about 198 kg of eggs annually, despite being the eleventh-largest egg producer in the world .
Either during production or consumption, metals can taint eggs.
Eggs may become tainted with heavy metals through chicken feed and drinking water, both of which are largely influenced by the environment. Heavy metals have several acute and long-term harmful effects on different human organs. Examples of the adverse effects of heavy metal toxicity include cancer, gastrointestinal and renal dysfunction, nervous system diseases, skin lesions, vascular damage, immune system malfunction, and birth abnormalities (Balali-Mood et al., 2021;Johri et al., 2010).
Heavy metal contamination of eggs is a significant problem for public health. Long-term exposure to even small doses may cause anxiety, depression, restlessness, hypertension, anemia, damage to the fetal brain, tremors, lungs, liver, kidney diseases, and autoimmunity diseases, such as rheumatoid arthritis (Aliu et al., 2021;Järup, 2003). According to studies, low-dose exposure to Cd, and Pb, interactions in mixtures showed higher levels of toxicity than individual heavy metals (Cobbina et al., 2015). Exposure to Pb has been linked to gastrointestinal colitis, brain function disorders, anemia, leukemia, hyperactivity, and thrombotic diseases (Abedi et al., 2020). Additionally, substances containing cadmium and lead were identified as carcinogens to humans. Exposure to Cd is related to painful diseases known as Itai-Itai. Hypercalciuria, inconsistent calcium metabolism, hypertension in expectant women, kidney stone formation, and bone demineralization are some further negative impacts of Cd on human health. (Satarug et al., 2017). Due to the extensive use of pesticides, municipal wastewater, industrial effluents, and raw sewage for irrigation, both necessary and nonessential components are continually replenishing our food chain (Mitra et al., 2022). One of the major problems posed by heavy metals is that they are not usually metabolized by the body (Wang et al., 2017). Tehran, the capital of Iran, has received a reputation as one of the world's most polluted cities in recent years (Goharipour & Firoozabadi, 2022). This pollution can affect human health directly or indirectly through contamination of the food chain. The food system depends heavily on chickens, and their contamination can have disastrous effects. Therefore, this study was conducted with a special look at hen eggs as one of the most consumed foods in the food basket of most low-income Iranian households. Pb and Cd concentrations, which are the two most hazardous heavy metals due to their cumulatively harmful influence on human health, were used to evaluate the quality of eggs to determine their potential risk to human health. Because hen eggs are one of the most popular foods in the food baskets of the majority of low-income Iranian households. Egg quality was assessed to establish the possible risk to human health using Pb and Cd concentrations, the two most dangerous heavy metals due to their cumulatively negative impact on human health.

| Samples
Forty-two egg samples were selected from 21 best-selling brands of supermarkets in Tehran. All samples (two eggs from each batch content of 12 eggs) were first visually inspected by candling and only fresh and unscathed eggs (no cracks, visibly clean eggs, and pin-holes) were chosen. Then, they were cleaned with distilled water and after coding and transferred to the laboratory for chemical analysis. There, samples were immediately refrigerated at 4°C before the preparation process. Instead of using metal tools, chemically stable sterile flacon tube tools were utilized to prevent any chemical contamination. The preparation for the analytical work was done right away. The entire piece of equipment was cleaned with diluted Nitric acid (HNO 3 10%) and then distilled water before use to prevent element contamination (Meermann & Nischwitz, 2018).

| Samples preparation
Before being weighed with a computerized analytical balance that had a 0.0001 g precision, all samples were thoroughly cleaned in deionized water. The eggshell is then gently separated from the egg's contents. The next step involved carefully mixing and homogenizing each complete egg product before pouring it into Petri plates to dry for 24 h at 70°C. addition, this finding provides accurate and reliable information for policymakers to improve food safety status to reduce public health hazards.

K E Y W O R D S
cadmium, health risk assessment, heavy metal, hen egg, lead, simulation To digest half a gram of dried egg samples, 10 mL of 70% nitric acid and 30% hydrogen peroxide (v/v) were purchased from Merk (Darmstadt, Germany) and left at room temperature for one night.
Until the solution was clear, the digestion was finished at 150°C for 4 h after the solution had been cooled to room temperature (22-23°C), it was diluted to 50 mL with deiodine water and then filtered through 0.45 L of acid-resistant filter paper. For further analysis, the solution was kept at 4°C. The liquefied solution was filtrated and diluted with 20% HNO 3 before being analyzed by inductively coupled plasma mass spectrometry (ICP-MS, ULTIMA2, 6100 DRC-e Perkin Elmer Elan). The following steps were done: washing glassware containers used for analysis with detergent and rinsing several times with tap water, soaking overnight in 6 N HNO 3 (Merk) solutions, and finally rinsing with deionized water.
According to the FDA Elemental Analysis Manual, samples were examined for total Pb and Cd using heat-block-assisted acid digestion and the ICP-MS technique. The blank solution was made in the same way but without an egg. In Table 1, the ICP-MS conditions for detecting lead and cadmium in eggs are listed ICP-MS.
Ionizing the sample with an inductively coupled plasma is the method used in inductively coupled plasma mass spectrometry or ICP-MS. It is well known for its application in identifying a variety of non-metals and metals in liquid samples at incredibly low concentrations. It is well known that this method is the most rapid and accurate for detecting heavy metal concentration in the food industry (Meermann & Nischwitz, 2018).

| Estimate daily intake
The average body weight (BW) of adult consumers, the mean levels of these metals in eggs, and the number of eggs ingested were used to compute the Estimate daily intake (EDI) of Pb and Cd.
The EDI of Pb and Cd was estimated according to Equation 1 (Dadar et al., 2017).
Estimate daily intake is estimated daily intake (μg kg −1 BW/ day); F IR , daily eggs consumption (ml day −1 ); C M , mean level of metal (mg mL −1 ); and W AB , and average body weight (kg). According

| Non-carcinogenic risk estimation
The non-carcinogenic danger to egg consumers was assessed using the target hazard quotient (THQ) of Pb and Cd from hen eggs (EPA, 2004;Rahmani et al., 2018)

by equation 2:
The frequency of exposure (EF) and exposure time (EP) deemed necessary to achieve the Iranian mean longevity were 365 days annually and 70 years, respectively. Pb and Cd had oral reference doses (RFD) of 0.004 and 0.001, respectively. For non-carcinogens, the TA was 25,550 days (EF × ED).

| Carcinogenic risk estimation
The incremental lifetime cancer risk (ILCR) was estimated to determine the possibility of cancer risk of Pb in adults through consuming eggs (Abedi et al., 2020) and calculated by equation 3: The cancer slope factor (CSF) is the risk produced by a lifetime mean dose of 1 mg kg −1 BW/day . CSF for Pb was 0.0085 mg kg −1 day (OEHHA, 2009). (1)

| Monte Carlo simulation (MCS) technique
Estimating health risks can involve some uncertainty (Chen et al., 2012). High uncertainty is seen when using single-point measurements to determine health risks associated with exposure to contaminants such as hazardous metals. To reduce the uncertainties in the health risk assessment, MCS was employed in our investigation as a probabilistic technique Ru et al., 2013).
The creation of risk assessment models was done using the Crystal Ball software (version 11.1.2.4, Oracle, Inc., USA). The criteria for endangered exposed populations is a percentile of 95% of THQ and ILCR in the cumulative probability graph, and there were 1000 repeats. (Qu et al., 2012).

| Statistical analysis
Data analysis was carried out by the SPSS software (SPSS Inc., version 16, Chicago, IL, USA) following the collection of the required data.
For categorical variables, data were summarized using frequency (%), mean (±Standard Error), and median (minimum-maximum) for both normal and non-normal distributions. The data's normality was examined using the Kolmogorov-Smirnov test. Kruskal-Wallis and Spearman's correlation matrix analyses were used to compare two groups against one another. p-values <.05 were considered statistically significant.
The average of two measurements, with repeatability of under 10%, was used to determine the trace element concentrations in each sample. The results were given in milligrams per kilogram of the sample's moist weight. For the statistical analysis, it was assumed that the concentration was equal to LOQ/2 when the element's content was below the limit of quantification (LOQ) (Esposito et al., 2016).

| RE SULTS AND D ISCUSS I ON
Heavy metal exposure has grown as a result of environmental, industrial, and agricultural activity as well as modern industrialization, all of which have negative impacts on human health. The environmental problem of hazardous metal contamination of water and air affects hundreds of millions of people worldwide. Heavy metal pollution in food is a problem for both human and animal health. In this context, the concentration of heavy metals in food, air, and water resources is evaluated . The hazardous metal lead has a significant negative influence on individuals and communities.
It can impact psychological and behavioral functions when present in low concentrations, and it can be harmful when present in high quantities (Tong et al., 2000). Exposure to Pb may raise the risk of stomach, kidney, lung, and brain cancer in both men and women .
In nature, lead is a nonbiodegradable metal that occurs in quite small concentrations. Human activities including manufacturing, mining, and the combustion of fossil fuels are causing atmospheric lead levels to constantly rise. When exposure levels are higher than what is considered safe, lead is harmful to humans. Children are more likely to become poisoned by lead than adults, and the severity of the poisoning rises when they come into touch with dust that contains environmental lead . And also, cadmium is a toxicant and carcinogenic metal. In addition to its carcinogenic  (Kabeer et al., 2021;ul Islam et al., 2014). The correlation between the Pb and Cd was significant at the 0.05 level (r = 0.350) in Table 2.

| Health risk assessment
In addition to estimating the overall concentration of lead and cadmium by comparing the standard permissible limits, other parameters like exposure time, per capita intake, metal toxicity, and body weight are highly useful in determining the potential health risk.
Therefore, exposure assessments as well as non-carcinogenic and carcinogenic risk assessments for adults were carried out using accurate data interpretation Rahmani et al., 2018).

| Exposure assessment
Estimate daily intake, EWI, and EMI were calculated using two scenarios for the total and maximum concentrations of these metals, and the findings were compared with the JECFA-set provisional acceptable weekly intake (PTWI) and provisional tolerable monthly intake (PTMI); it was possible to estimate the dietary exposure to Pb and Cd from eating eggs. Table 4  Regarding the overall concentration of Pb in eggs in the present study (0.007 mg kg −l ), the weekly intake of Pb for adults through eggs consumption was estimated to be 0.014 mg/week which was higher than those reported by (Zheng et al., 2007; (Farahani et al., 2015), which seems that compared with the data of the current study, there has been a decreasing trend in Iran (Table 4).
Due to its long half-life, the JECFA declared in 2010 that daily cadmium ingestion through food consumption has very little impact on overall exposure. As a result, the acceptable and dietary intake of Cd should be assessed over a month or several months, respectively,  When a metal's THQ is greater than 1, negative effects are possible; however, when is less than 1, detrimental effects are unlikely to occur (Dadar et al., 2017;Fakhri et al., 2018;Rahmani et al., 2018).  (Table 5).

| Non-carcinogenic risk evaluation
Lead builds up over time in bones and soft tissue, but more significantly, its presence in blood and bones over its half-life has been demonstrated. The cardiovascular effects and developmental neurotoxicity in adults and young children were acknowledged by the JECFA and CONTAM Panel of the EFSA (European Food Safety Authority) as being significant for the assessment of lead risk (Abadin et al., 2007;Norouzirad et al., 2018). Due to an interaction with a protein with a high sulfhydryl content, the majority of the absorbed Cd is stored in the kidneys. The half-life of Cd in human kidneys could be up to 30 years. Emphysema, chronic obstructive lung disease, and chronic renal tubular disease are the main outcomes of a long-term cadmium buildup, particularly in children (Faroon et al., 2012;Friberg, 2017). Adult THQ values from consuming eggs have been reported by several authors and are displayed in Table 5.
For example (Gonzalez et al., 2017) reported values of 0.035 for Pb, which is equal to that discovered in this study, and 0.045 for Cd, which is greater than that obtained in this work. These results were published in Mexico. Pb and Cd concentrations were discovered by (Zheng et al., 2007)  It is important to note that exposure to these toxic metals from sources other than those examined in the study, such as water, skin contact, inhalation of dust, and consumption of other foods, may result in THQ values greater than 1, which would indicate a significant risk to the health of the exposed population.

| Carcinogenic risk
Numerous studies have demonstrated that exposure to environmental contaminants, such as toxic elements, increases the risk of cancer, although a range of factors, including age, race, and gender, may contribute to the development of cancer (Antwi et al., 2015;Farokhi et al., 2017;Steenland & Boffetta, 2000).
IARC has identified Pb and Cd as possible contributors to the development of human cancer, as was previously mentioned (IARC, 1993(IARC, , 2006(IARC, , 2017. To calculate the carcinogenic risk for adult egg users in the current investigation, the ILCR value was used. Because the CSF for cadmium's risk of oral cancer has not yet been established, we only estimated the ILCR for lead. (Figure 3).
Incremental lifetime cancer risks for Pb were found to be 2.2E-05 and 2.8E-05, respectively. The U.S. Environmental Protection Agency (US-EPA) states that the safe limit for cancer risk is below approximately 1 chance in 1000,000 lifetime exposure (ILCR < 10 −6 ), the threshold risk limit (ILCR > 10 −4 ) for a chance of cancer is above 1 in 10,000 exposure, where corrective measures are significant, and the moderate risk level (ILCR > 10 −3 ) is above 1 in 1000 where public health safety assessment is more critical (Pepper et al., 2012;Tchounwou et al., 2012). The findings showed that the adult group's consumption of eggs did not increase their risk of developing cancer and that their health is almost safe at this point (10 −4 > ILCR < 10 −6 ).
When calculating health hazards, there may be some uncertainty (Chen et al., 2012). High uncertainty is seen when single-point data are used to determine health risks associated with exposure to con- Our food chain is constantly being replenished with essential and non-essential materials as a result of the excessive use of agrochemicals, municipal wastewater, industrial effluents, and raw sewage for (Tongesayi et al., 2013).

| Limitation
It is recommended that the international establishes this restric- reprehensive of all eggs in Iran. Moreover, this study represents the heavy metals content in eggs and does not necessarily reflect other product consumption.

| CON CLUS ION
An appropriate and sensitive ICP-MS method was used to evaluate the levels of lead and cadmium in hen egg samples in Iran. The findings of the present investigation showed that lead and cadmium levels in all evaluated eggs were suitable for human consumption.
Adults' Pb and Cd exposure from eating eggs was significantly lower than the risk levels established by JECFA, per the exposure assessment. According to the THQ values of these dangerous metals, which were below one value, egg eating by Iranian consumers does not present a non-carcinogenic risk. Due to the Pb concentration in eggs, the carcinogenic risk assessment concluded that adults in Iran are not at increased risk of developing cancer (ILCR > 10 −6 ).
However, other meals containing these heavy metals can also introduce lead and cadmium into the body. As a result, it is advised that lead and cadmium levels in hen eggs and other foods in Iran be routinely monitored.

ACK N OWLED G M ENTS
The

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no conflicts of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data will be available upon request from the corresponding authors.

E TH I C A L A PPROVA L
The Ethics Committee of the National Nutrition and Food