Analysis of genotoxic effects of food preservatives sodium acetate (E262) and sodium sulfite (E221) in human lymphocytes

Abstract

Food preservatives are a large class of food additives that generally prevent microbiological spoilage. Sodium acetate (SA) and sodium sulfite (SS) are food preservatives, and the aim of this study was to investigate their genotoxic and cytotoxic effects. The genotoxic effects of SA and SS were examined by chromosomal aberrations (CA) and micronucleus (MN) assays in human lymphocytes in vitro. In addition, the effects of these two preservative additives on mitotic index (MI) and nuclear division index (NDI) were also investigated. SA and SS significantly induced CAs and MN frequencies and caused a decrease in MI especially at higher concentrations. Neither food preservative caused any change in the NDI. In the light of the data obtained, it was concluded that SA and SS may have cytotoxic and genotoxic effects on human lymphocytes, especially at high concentrations. Therefore, their use at lower concentrations, which may be safer, should be encouraged.

Introduction

Food additives (FAs) are one of the most prominent groups of chemicals preferred for different purposes. FAs are generally defined as "substances used to affect the properties of food in the desired way". They are substances used to maintain and increase the structural properties of foods such as taste, smell, flavor and appearance from the production stage to the consumption stage (Altunkaynak and Avuloglu-Yilmaz, 2021; Honma et al., 2022). Food preservatives are the most used group of FAs. Food preservatives reduce economic concerns by preserving the quality and flavor of food and extending its shelf life. Food preservatives added to foods slow down spoilage caused by mold, air, fungi, bacteria, and yeasts. They prevent changes in the taste of food, unwanted odor formation and enzymatic browning. Sodium acetate (E 262) (SA) and sodium sulfite (E 221) (SS) are frequently used food preservatives. SA is in the form of the sodium salt of acetic acid and is anhydrous. According to the Turkish Food Codex, SA is allowed to be used in meat mixtures prepared from minced meat, prepackaged, and prepared meat mixtures to which additives or other ingredients other than salt are added (Turkish Food Codex, 2013). SS is in the form of sodium salt of sulfuric acid. SS is a preservative food additive that prevents food spoilage and discoloration, which is as good as the lowest bleaching and oxidizing factors. It masks bacterial spoilage characterized by discoloration. Although it can prevent browning in meat, it is not allowed in meat products. It is used as a preservative in products containing egg yolk and in foods such as beer, salads, caramel, and bread (Benli and Turkoglu, 2017).

Genotoxicity is a general term covering damage to genetic material by physical or chemical agents (mutagens and carcinogens). Genotoxic substances that interact with enzymes that allow the genome or DNA to be copied and cause mutation, damage or cause some changes in DNA are referred to as genotoxic effects (Andarza et al., 2022; Menz et al., 2023).

The increase in various health problems caused by agents with genotoxic effects has led to the development of short-term genotoxicity tests to detect these agents. Short-term genotoxicity tests have become the most preferred tests for the detection of carcinogenic and genotoxic potentials of FAs with their validity accepted all over the world. Among these genotoxicity tests, the most used tests are chromosomal aberrations (CAs) and micronucleus (MN) tests (Yuzbasioglu and Avuloglu-Yilmaz, 2022). These tests are validated, internationally recognized methods that the OECD considers appropriate for testing chemicals for genotoxicity CA and MN tests are complementary assays. The CA test identifies structural and numerical changes in chromosomes. MNs are small nuclei separate from the main nucleus and are the result of clastagenic and/or anogenic effects (OECD 2016a, b).

The aim of this study was to investigate the genotoxic effects of the food preservatives SA and SS in human peripheral lymphocyte culture by CA and MN tests, which are frequently used in foods but have few studies on their genetic effects in human cells. It is thought that this research will be of great importance in updating the permits given or to be given for the use of these two preservatives and increasing the awareness of producers and consumers, on the other hand, it will be the basis for further research with SA and SS.

The European Union regulates the field of food safety through various regulations, directives and decisions. These also focus on agricultural nutrition and consumer protection (Bondoc, 2015a, 2016a). European regulations cover many sub-areas, such as the authorisation and licensing of food additives and their traceability in the product chain, the suspension of the use of certain additives, as well as food safety and the protection of consumer health and interests (Bondoc 2015b, 2016b). European Food Safety Authority (EFSA) was established by the European Union and is responsible for contributing to the safety of the food chain, assessing risks in scientific terms, and sharing the results of risk assessment with the public. Importantly, reassessment report published in 2022, EFSA concluded that sulphites (including SS) may not be safe, especially for those who consume them at high levels. It also noted that there are gaps in toxicity data and that it is not known exactly how this may affect health. It is clear that this study will contribute to such data. In addition, EFSA announced in the same year that it would re-evaluate acetates used as food additives, including in SA. It is possible that this study will contribute to this evaluation (EFSA, 2022a, b).

Materials and methods

Materials

In this study, the genotoxic and cytotoxic effects of SA and SS, which are among the preservatives of food additives, were investigated in human peripheral blood lymphocytes in vitro. For this purpose, peripheral blood was obtained from two healthy female donors. The preferred characteristics of the donors included in the study were not having any health problems, not consuming drugs, smoking, alcohol, and being between 22–27 years of age. This study was conducted with the permission of Amasya University Ethics Committee numbered 03.06.2021-87.

Commercial forms of SA (CAS No: 127-09-3 Sigma-Aldrich and SS (CAS No: 7757-83-7 Sigma-Aldrich) were used and their purity was ≥ 0.98%. The genotoxic effects of the preservatives SA and SS were determined in cultured human peripheral lymphocytes using two different genotoxicity tests. These tests are CA and MN assays. In addition, the effects of these chemicals on MI and NDI were also evaluated.

Concentration selection

According to JECFA and the Turkish Food Codex, the ADI (ADI Not Specified) of SA and SS were determined (Turkish Food Codex, 2013). Therefore, the preferred amounts in humans are not clear. In this study, the LD50 dose of these substances in the literature was used as a reference to determine the preferred concentrations in the two tests. Using these doses, preliminary studies were carried out to determine the concentrations to be preferred in this study. SA concentrations of 3.91; 7.81; 15.63; 31.25; 62.50; 125.00; 250.00 and 500.00 µg/mL were tested and according to the results, concentrations of 15.63; 31.25; 62.50; 125.00 and 250.00 µg/mL were preferred. All concentrations tested in the preliminary studies of SS were 3.91; 7.81; 15.63; 31.25; 62.5; 125.00 and 250.00 µg/mL, of which concentrations of 3.91; 7.81; 15.63; 31.25 and 62.50 µg/mL were preferred. In preliminary studies, MI values were determined to determine the concentrations to be used. MI is a useful biomarker for evaluating the cytotoxic and cytostatic activities of chemicals in tumoral and normal experimental models and measures the proportion of cells undergoing mitosis.

CA assay

CA assay was performed using the method of Evans (1984) with some modifications (Evans, 1984). 0.2 mL of heparinized blood samples from donors were added to Lymphoplus medium and incubated at 37 °C for 72 h. At the end of the 24th and 48th hour from the beginning of the culture, the concentrations of SA and SS given above were applied to the cells. In addition, distilled water was used as a negative control and MMC (Mitomycin-C) at 0.20 µg/mL was used as a positive control. All procedures for cell harvesting preparation and staining were performed as described in Yuzbasioglu et al. (2006). For each concentration, a total of 400 metaphases with well-dispersed chromosomes (200 metaphases per donor) were evaluated. The percentage of abnormal cells and the number of chromosomal abnormalities per cell were determined. For MI, a total of 4000 cells were analyzed, 2000 cells per donor. The ratio of the number of dividing cells to the total number of cells was calculated as a percentage.

MN assay

The method of Fenech (2000) was used with some modifications in the application of the MN test (Fenech, 2000). In order to culture human lymphocytes, 0.2 mL of blood was added to culture tubes containing Lymphoplus medium. All tubes were incubated at 37 °C for 72 h. Cells were treated with concentrations of SA and SS for 48 h. Negative (distilled water) and positive control (MMC, 0.20 µg/mL) were added to each set of experiments. To inhibit cytokinesis, 7 µg/mL of Cytochalasin B (Cyt B) was added to all culture tubes at 44 h of culture. Preparation, staining and counting of preparations were as described in Yuzbasioglu et al. (2006).

Statistical analysis

In this study, evaluation of CA and MN assays and MI, NBI findings, the results obtained from the control and treatment concentrations groups were evaluated by z test. On the other hand, regression analysis was performed with SPSS 22.0 programme to reveal the concentration-effect relationship for the treatment groups which showed significant variability compared to control concentrations.

Results and discussion

Seven types of chromosomal aberrations were observed in the CA assay performed by SA treatment of in vitro human peripheral lymphocytes for 24 and 48 h. The frequency of these aberrations was shown in Table 1. As a results, SA increased the aberrations per cell (CA/cell) and abnormal cell frequency in a concentration-dependent manner compared to the control at all treatment periods (for 24-h r = 0.96, for 48-h r = 0.85). This increase was significant at the four highest concentrations (31.25; 62.50; 125.00 and 250.00 μg/mL) in the 24-h treatment and at the highest concentration (250.00 μg/mL) in the 48-h treatment (Table 1).

Table 1.

CAs in peripheral human lymphocytes induced by SA

Test substance	Treatment		Abnormalities							Abnormal cell  ± SE (%)	CA/cell ± SE
	Period (hour)	Concentration (μg/mL)	ctb	csb	scu	dic	cte	f	p
Control	24	0.00	1	–	1	–	–	1	–	0.75 ± 0.43	0.0075 ± 0.0043
MMC	24	0.20	7	8	16	3	1	7	1	10.75 ± 1.55	0.1075 ± 0.0155
SA	24	15.63	2	1	2	–	–	3	–	2.00 ± 0.70	0.0200 ± 0.0070
		31.25	3	3	9	–	1	1	–	4.25 ± 1.01**	0.0425 ± 0.0101**
		62.50	4	4	6	–	–	6	1	5.25 ± 1.12**	0.0525 ± 0.0112**
		125.00	8	3	7	–	–	3	–	5.25 ± 1.12**	0.0525 ± 0.0112**
		250.00	5	7	4	–	2	9	–	6.75 ± 1.25***	0.0675 ± 0.0125***
Control	48	0.00	–	3	1	–	–	3	–	1.75 ± 0.66	0.0175 ± 0.0066
MMC	48	0.20	13	9	10	5	2	12	1	13.00 ± 1.68	0.1300 ± 0.0168
SA	48	15.63	2	5	3	–	–	1	–	2.75 ± 0.82	0.0275 ± 0.0082
		31.25	–	–	2	–	4	4	1	2.75 ± 0.82	0.0275 ± 0.0082
		62.50	2	1	1	–	2	1	–	1.75 ± 0.66	0.0175 ± 0.0066
		125.00	1	7	6	–	1	–	–	3.75 ± 0.95	0.0375 ± 0.0095
		250.00	5	–	9	–	5	3	–	5.50 ± 1.14**	0.0550 ± 0.0114**
Frequency of abnormalities (%)
			20.00	19.37	30.63	0.00	9.38	19.37	1.25

ctb chromatid break, csb chromosome break, scu sister chromatid union, dic dicentric chromosome, cte chromatid exchange, f fragment, p polyploidy

^**Significantly different from the control p < 0.01 (z-test)

^***Significantly different from the control p < 0.001 (z-test)

On the other hand, MI decreased significantly at concentrations of 15.63, 62.50 and 250.00 µg/mL in the 24 h treatment compared to the control. In the 48-h treatment, a significant decrease was observed in the three highest concentrations (62.50; 125.00 and 250.00 µg/mL) (Fig. 1). However, this decrease was weakly correlated with concentration in the 24 h (r = -0.59) and not correlated with concentration in the 48 h treatment (r = -0.36). SA treatment was caused increase in the percentage of binuclear cells carrying micronuclei in human lymphocytes. This increase was statistically significant (except for the concentrations of 15.63 µg/mL and 31.25 µg/mL) and concentration dependent (r = 0.98) (Fig. 2). Additionally, SA did not affect the NDI (Fig. 3).

Fig. 1.

Effect of SA treatment on MI frequencies in peripheral human lymphocytes

Fig. 2.

Effect of SA treatment on MN frequencies in peripheral human lymphocytes

Fig. 3.

Effect of SA treatment on NDI in peripheral human lymphocytes

Table 2 shows the types of abnormalities, abnormal cell frequencies and ratios of abnormalities per cell observed at all concentrations after 24 and 48 h of SS treatment with peripheral human blood lymphocytes. SS treatment of peripheral human lymphocyte culture for 24 h and 48 h resulted in a statistically significant increase in abnormal cell percentage and abnormality frequency per cell at all treatment times and concentrations compared to control (Table 2). These increases were concentration dependent (24 h r = 0.82, 48 h r = 0.88 for abnormal cell percentage and aberrations per cell).

Table 2.

CAs in peripheral human lymphocytes induced by SS

Test substance	Treatment		Abnormalities							Abnormal cell ± SE (%)	CA/cell ± SE
	Period (hour	Concentration (μg/mL)	ctb	csb	scu	dic	cte	f	p
Control	24	0.00	–	–	2	–	–	1	–	0.75 ± 0.43	0.0075 ± 0.0043
MMC	24	0.20	6	2	5	2	3	16	1	8.75 ± 1.41	0.0875 ± 0.0141
SS	24	3.91	8	–	4	1	–	2	–	3.75 ± 0.95**	0.0375 ± 0.0095**
		7.81	5	2	6	–	–	1	–	3.50 ± 0.92**	0.0350 ± 0.0092**
		15.63	–	1	10	2	2	3	–	4.50 ± 1.04***	0.0450 ± 0.0104***
		31.25	–	6	2	1	4	6	1	5.00 ± 1.09***	0.0500 ± 0.0109***
		62.50	6	3	5	–	2	3	–	4.75 ± 1.06***	0.0475 ± 0.0106***
Control	48	0.00	4	1	3	–	–	2	–	2.50 ± 0.78	0.0250 ± 0.0078
MMC	48	0.20	3	3	34	–	–	23	1	16.00 ± 1.83	0.1600 ± 0.0199
SS	48	3.91	4	–	5	3	12	1	–	6.25 ± 1.21**	0.0625 ± 0.0121**
		7.81	6	3	6	2	7	6	–	7.50 ± 1.32**	0.0750 ± 0.0132**
		15.63	9	–	15	3	5	2	–	8.50 ± 1.39***	0.0850 ± 0.0139***
		31.25	6	4	10	1	9	3	–	8.25 ± 1.38***	0.0825 ± 0.0138***
		62.50	–	6	14	1	17	4	–	10.50 ± 1.53***	0.1050 ± 0.0153***
Frequency of abnormalities (%)
			17.60	10.00	30.80	5.60	23.20	12.40	0.40

ctb chromatid break, csb chromosome break, scu sister chromatid union, dic dicentric chromosome, cte chromatid exchange, f fragment, p polyploidy

^**Significantly different from the control p < 0.01 (z-test)

^***Significantly different from the control p < 0.001 (z-test)

SS significantly decreased MI compared to the control at all treatment times and concentrations (except the concentration of 3.91 μg/mL in the 24 h treatment) (Fig. 4). These decreases were not correlated with concentrations in the 24 h treatment (r = -0.39), and weakly correlated with concentration in the 48-h treatment (r = -0.60). SS treatment increased the percentage of binucleate with MN in the culture. The increase was statistically significant compared to the control (except for the concentration of 3.91 µg/mL) and was concentration dependent (r = 0.85) (Fig. 5). On the hand, SS did not affect the NDI (Fig. 6).

Fig. 4.

Effect of SS treatment on MI frequencies in peripheral human lymphocytes

Fig. 5.

Effect of SS treatment on MN frequencies in peripheral human lymphocytes

Fig. 6.

Effect of SS treatment on NDI in peripheral human lymphocytes

It is one of the well-known facts that food is a vital need for humans, which is indispensable and cannot be postponed. However, foods offered for consumption in relation to developing science, technology and industry contain more than one chemical substance. Nowadays, as people tend to more practical foods and ready-made foods, the use of food preservatives has increased in order to extend the shelf life of the food. Production and consumption relations of foods, the use of food preservatives have become essential for today's living conditions. However, since studies have started to show that preservatives can pose genotoxic and carcinogenic risks in various organisms, research on these issues has increased and restrictions have been placed on the use of food additives (Altunkaynak and Yılmaz, 2021; Thomas et al., 2023).

In general, it is revealed that even the little used values of different chemical agents, whose usage amounts are increasing over time, can be teratogenic, carcinogenic and mutagenic. For this reason, it is important to determine whether physical and chemical agents with the potential to have these effects have genotoxic, mutagenic and carcinogenic effects for the human genome. Positive results in mutagenicity tests reveal that more than one genotoxic substance is also carcinogenic (Al-Khdour et al., 2023; Menz et al., 2023).

Food additives undergo safety tests, including genotoxicity, before being placed on the market, as is the case for many other chemicals. The recommended genotoxicity tests of these substances are as follows; Ames, in vitro CA assay with mammalian cells and in vivo CA assay with mammalian hematopoietic cells (FDA, 2007). Besides these, it is recommended to evaluate with different cell groups and test systems. Apart from all these, national and international authorities emphasise the necessity of re-evaluation of food additives while they are still in use and carry out studies accordingly. EFSA’s expert Panel on Food Additives and Flavourings (FAF) is responsible for assessing the safety of new food additives as well as conducting re-evaluations of all food additives authorised for use in the EU before 2009 (EC 1333/2008). The Committee evaluates the studies performed, monitors developments, discusses possible future situations and, most importantly, draws attention to food additives for which data are lacking and calls for studies to be carried out. In this context, both these organisations and independent researchers conduct tests on these issues. The cytotoxicity and genotoxicity studies with SA and SS have been carried out in different cell groups, but there are no studies in human lymphocytes.

On the other hand, there are some limitations of the test systems used in this study. Since all tests were performed under in vitro conditions and without the use of metabolic activation, they do not fully represent in vivo conditions, but the results were supported by two different tests. In addition, as stated by the OECD, conditions such as excessive cytotoxicity levels and pH levels of the medium may lead to false results such as chromosome damage or MN formation that are not caused by direct interaction between the test chemicals and chromosomes. Therefore, preliminary concentration experiments were carried out and the culture medium was also controlled in these experiments. In the MN test, one of the limitations stated by OECD in the procedure is that mitosis must have occurred in both treated and untreated cultures to evaluate MN formation. This was overcome by inhibiting cytokinesis at the 44th hour of the 72-h culture and performing this analysis in cells that underwent at least one mitosis after chemical treatment. A similar situation was also reported for the CA assay. In order to detect chromosomal aberrations that may result from the clastogenic effect, cells in metaphase must be used. Therefore, cells at metaphase were evaluated for aberrations in both treated and untreated cultures (OECD 2016a, b).

In this study, food preservatives SA and SS induced CA and MN formation, decreased MI and did not affect NDI in human lymphocytes in vitro. There are different genotoxicity studies with SA and SS. In a study conducted in 2018, it was shown by MTT assay that cell viability decreased in SA-treated HUVEC cells depending on time and dose. In the same study, it did not cause fragmentation of genomic material in DAPI-staining and DNA ladder tests. Therefore, it was stated that SA may not be cytogenotoxic at low doses and it would be appropriate to use it at low doses (Mohammadzadeh-Aghdash et al., 2018). Similar results were obtained in this study and SA showed genotoxic effect at high concentrations in human lymphocytes. According to different study, the genotoxic and cytotoxic effect of SA was investigated in the lymphocytes of adult male Sprague–Dawley rats by comet, MTT and LDH (lactate dehydrogenase) tests. SA was applied to lymphocytes at concentrations of 50, 100 and 200 mM. According to the results of the comet assay, it was observed that SA increased the tail length, density and moment depending on the concentration. In addition, a concentration-dependent decrease in both cell viability and proliferation was detected in the MTT test, and a concentration-dependent increase in LDH release was reported. Considering all the results, it was determined that SA was cytotoxic and genotoxic at the investigated concentrations (Abd-Elhakim et al., 2018). The fact that SA showed both genotoxic and cytotoxic effects in human lymphocytes in this study also supports these results.

According to the EFSA report published in 2022, SS did not cause a mutagenic effect in the Ames test (Ishidate et al., 1984) but gave a positive finding in a spore rec test using Bacillus subtilis strain M45 (Ueno et al., 2002). In in vitro mammalian cells, SS showed no clastogenic activity in the in vitro CAs assay (Ishidate et al., 1984) but induced gene mutation at the gpt locus in AS52 cells (Meng and Zhang, 1999), which was associated with cytotoxicity at the highest concentration used. SS did not induce MN in mouse bone marrow but was shown to cause DNA damage in vivo in different organs of mice in the alkaline comet assay (Meng et al., 2004). In mitogen-stimulated peripheral mononuclear cells isolated from blood from healthy donors, 1 mM SS was shown to have a suppressive effect on blood cells (Winkler et al., 2006). In a recent study, the genotoxic effect of SS was investigated in Drosophila melanogaster by SMART and comet assay. Two different Drosophila strains, a wild type and a strain carrying the wtsMT4-1 (a lethal allele) allele, were used. The animals in the experimental group were given 100 mM SS. According to the results of the SMART assay, tumor induction and frequency were determined with a high level of somatic mutation compared to the negative control. According to the comet assay findings, a significant increase in DNA damage was also observed, and tail length, density and moment were induced statistically significantly. According to the findings, it was emphasized that SS was genotoxic, and the results were a clear indicator of DNA degradation and chain breaks (El-Hefny et al., 2021). Similarly, the genotoxic and cytotoxic effect of SS in human peripheral lymphocytes in this study agrees with the results of the study described above.

Under in vivo and in vitro conditions, the most important cellular response due to chemical causes is the delay or inhibition of the cell cycle. MI is one of the important parameter tests accepted as an indicator of cytotoxicity. The cytotoxic level can be determined by the decrease in MI. Inhibition of the G2 phase, decrease in ATP level, defects in the energy production, inhibition of DNA synthesis or suppression of enzymes responsible for spindle formation are shown as the reasons for the decrease in the value of MI (Van’t Hof, 1968; Yuzbasioglu et al., 2014). The decreases in MI frequency observed in this study may be due to the inhibition of enzymes and/or proteins required for cell proliferation by SS and SA.

The toxic effect of SS and some other food additives on HepG2 cells and its mechanisms were investigated by CCK-8 assay. The results showed that SS decreased cell viability in a dose-dependent manner and induced DNA damage. In addition, it was reported that all doses significantly increased ROS levels compared to the control. This was accompanied by an increase in cell membrane permeability and intracellular calcium levels and a significant decrease in mitochondrial membrane potential in high dose groups. (Qu et al., 2017). On the other hand, Abd-Elhakim et al (2018) reported that the release of ROS by acetate may be a mechanism underlying the genotoxic and cytotoxic effects of SA in in vivo. Thus, the geno-cytotoxicity induced by SA and SS in this study, especially at high concentrations, may be due to high ROS levels. In the body, oxygen radicals and antioxidant defense mechanisms work in complete balance. During normal metabolism, the balance between ROS and antioxidants can be shifted towards ROS by some environmental prooxidants. This leads to lipid peroxidation, protein oxidation, DNA mutation or breakage, enzyme activation or inactivation. DNA damage, cell cycle dysregulation, DNA repair or replication disorder and gene mutations can be observed in cells after exposure to ROS. DNA is constantly damaged by endogenic and exogenic mutagens and carcinogens. These damages can be repaired by various mechanisms (Goode et al., 2002). Cells with unrepaired DNA damage progress towards apoptosis or malignant tumors. A deformation or reduced efficiency of DNA damage repair plays an important role in the development of cancer risk (Goode et al., 2002).

Studies have shown that defects in the function of proteins such as XRCC1, PARP-1 and DNA LIG3 cause single chain breaks in DNA (Wang et al., 2005). XRCC1 protein has a great importance in the repair of fragmented lesions and minor DNA damage. PARP-1 enzyme is responsible for DNA repair and is a nuclear enzyme that causes apoptosis and necrosis when overactivated (Roy and Cardone, 2002). According to the threshold level of PARP1 activity, DNA repair, apoptosis or necrosis occurs (Cory and Adams, 2002). The release of ROS that may be caused by the two preservative food additives used in this study may also cause disruptions in the functions of these proteins, causing single chain breaks in DNA and thus mutagenic effects. However, detailed studies on their mechanism of action are required.

In this study, the genotoxic effects of SA and SS, which are preservatives frequently used in foods, on peripheral human blood lymphocyte culture were revealed firstly. It was concluded that they may have cytotoxic and genotoxic effects at high concentrations. On the other hand, although the results of many cyto-genotoxicity studies support each other, there are also contradictions due to different results as pointed out by EFSA. Therefore, this study fills this gap in the literature.

The genotoxic effect observed in few studies for both food preservatives support the results of this work (Abd-Elhakim et al., 2018; El-Hefny et al., 2021; Qu et al., 2017). When considered together with these studies, It is recommended to use SA and SS at lower concentrations that can be considered safer and to conduct studies with different cell groups and assays. In order to protect human health in the best way, it is extremely important to be considered as a system in the food production chain, to continuously control food safety, to take into account scientific evidence and all kinds of risk assessments in all measures taken in order to protect consumer interest.

Authors contribution

This paper was produced from Pınar Altunkaynak’s Master's thesis. Ece Avuloglu-Yilmaz was supervisor of Pınar Altunkaynak’s Master's thesis. All authors contributed to the study conception and design.

Declarations

Conflict of interest

The authors declare no competing interests.