Abstract
Today, ready-to-eat foods to which various additives are frequently added are widely consumed. Food colorings constitute an essential part of these additives. Carmoisine (E-122) and Ponceau 4R (E-124) are the most commonly used azo food colorings. This study aimed to investigate the in vitro genotoxic effects of these two food dyes in human peripheral lymphocytes using four different and complementary genotoxicity tests (chromosome aberrations (CAs), sister chromatid exchange (SCEs), cytokinesis block micronucleus cytome (CBMN-Cyt) and comet). When four different concentrations (37.5, 75, 150, and 300 μg/mL) of both food dyes were applied to lymphocytes for 24 and 48 h, it was observed that only the highest concentration significantly increased the frequencies of CA and SCE. The mitotic index (MI) decreased compared to the control at all concentrations except the lowest one in the 24-h treatment of Carmoisine and the two highest concentrations (150, 300 μg/mL) in the 48-h treatment. In the 24-h Ponceau 4R treatment, MI decreased compared to the control at all concentrations except the lowest and all concentrations in the 48-h treatment. In contrast, Carmoisine and Ponceau 4R did not affect MN frequency. In the comet test, Carmoisine increased tail length only at the highest concentration, and Ponceau 4R increased tail length at the two highest concentrations. Ponceau 4R also increased tail moment only at the highest concentration. When the results of these four in vitro genotoxicity tests were evaluated together, it was concluded that both food colors were genotoxic, especially at high concentrations, but not at low concentrations.
Keywords: Carmoisine, Ponceau 4R, genotoxicity, human lymphocytes, In vitro
Introduction
Azo dyes are compounds consisting of a diazotized amine coupled to an amine or a phenol and containing one or more azo bonds. Azo dye precursors are aromatic amines.1 They are preferred owing to their chemical versatility, providing a wide range of vibrant colors, low cost, easy availability, stable and consistent structure, and lack of off-flavors and odors.2 They are the world’s most widely used artificial coloring agents.2,3 Various pathways to toxic aromatic amines can metabolize azo dyes entering the human body through digestion. Enzymes such as azoreductases, nitroreductases produced by the gut microbiota, or xanthine oxidases and P450 cytochromes found in mammalian cell cytoplasm may play a role here. As a result, N-hydroxylamines are formed and can cause DNA damage. Therefore, the intestine is recognized as a possible target organ for the carcinogenicity of azo dyes.4–7
One of the most widely used azo food dyes is Carmoisine, also known as Azorubine (E-122), which is used to add red color to foods. People are most frequently exposed to Carmoisine when consuming products such as soft drinks, sauces, condiments, marinades, bakery products, and sweets, including flavored dairy products, chilies, pickles, relishes, chutney, and piccalilli.8 It has been linked to DNA damage and tumors in animals. However, in 2009, an EFSA panel concluded that the potential genotoxicity was negligible and also stated that no evidence would force a change in the ADI (4 mg/kg bw per day).8,9 On the other hand, EFSA reduced the ADI from 4 to 0.7 mg/kg bw/day in 2015, taking into account the results showing that Ponceau 4 R azo dye has a carcinogenic potential and the intake of Ponceau 4 R is above the ADI.10 The most common foods causing exposure to Ponceau 4R are non-alcoholic beverages and desserts, including flavored milk products, sauces, seasonings, pickles, piccalilli, relishes, and chutney.10 While both food colorings are used in many countries, they are restricted or banned in some countries.9 The preference for clean-labeled products with less environmental impact increases daily.11 Therefore, it is essential to understand the risks of consuming artificial azo dyes in food and evaluate studies on their genotoxic effects.
Chemical or physical agents can induce genotoxicity and can be used as an indirect marker of a person’s susceptibility to cancer. Genomic instability occurs when the cell becomes predisposed to accumulate genomic mutations and may initiate the process of carcinogenesis.12,13 In this study, the chromosome aberrations (CA), cytokinesis block micronucleus cytome (CBMN-Cyt), and Comet tests, which are among the most preferred and reliable genotoxicity tests, as well as the sister chromatid exchange (SCE) test, were performed in human peripheral lymphocytes in vitro. The reliability of genotoxicity assessment depends on various tests, each sensitive to different types of DNA damage. This is due to the impossibility of identifying the three main types of genetic damage (gene mutation, structural, and numerical chromosome mutations) with a single test with reliable results.14,15
Previous studies have shown that Carmoisine is not genotoxic in Ames tests in Salmonella typhimurium and Escherichia coli, does not generate chromosomal abnormalities in mice, and does not increase CA and SCE rates in Chinese hamster ovary cells.16–21 In previous studies, Ponceau 4R gave negative results16,19,22 in some Ames tests performed on S. typhimurium and E. coli strains but positive results23,24 in others. It has been reported to increase the rate of chromosomal abnormality and DNA damage in mice25,26 while not affecting micronucleus frequency.27 However, in 2022, published results revealed that it increased the frequency of MN in mice.7 In a 2011 study, the genotoxicity of Carmoisine and Ponceau 4R was investigated using the micronucleus test in human peripheral blood samples. A significant increase in MN frequency was observed compared to the control.28
Due to the conflicting results, this study was planned to obtain more detailed data on the genotoxic potential of these two food dyes and is the first study (except the MN test) in human lymphocytes.
Materials and methods
In this study, chromosomal abnormality (CA), sister chromatid exchange (SCE), and cytokinesis block micronucleus cytome (CBMN-Cyt) tests were performed on cultured human peripheral lymphocytes and a comet test on isolated lymphocytes. Peripheral blood was obtained from two female volunteer donors aged 18–27 yr who did not use drugs or smoke, had no chronic disease, and were not exposed to any genotoxic agent. This research has been carried out with the permission of the Gazi University, Faculty of Medicine, Ethics Committee for Clinical Research (03/29/2021–09).
Chemicals
E-122 Carmoisine or Azorubine’s name is Disodyum;4-hidroksi-3-[(4-sülfonatonaftalen-1-il) diazenil] naftalen-1-sülfonat (Fig. 1a). Molecular weight: 502.43 g/moL, molecular formula C20H12N2Na2O7S2, and CAS No: 3567-69-9 (Sigma-Aldrich).
Figure 1.
The structural formula of Carmoisine (a) and Ponceau 4R (b).
E-124 Ponceau 4R’s name is Trisodyum;7-hidroksi-8-[(4-sülfonatonaftalen-1-il) diazenil] naftalen-1,3-disülfonat (Fig. 1b). Molecular weight: 604.7 g/moL, molecular formula C20H11N2Na3O10S3, and CAS No: 2611-82-7 (Sigma-Aldrich).
Colchicine (CAS. No: 64–86-8), Bromodeoxyuridine (CAS No: 59–14-3), Cytochalasin B (CAS No: 14930–96-2), and Mitomycin C (CAS No: 50–07-7) were obtained from Sigma. LymphoPlus Medium (CAS No: CY100–100), DMSO (CAS. No: 67–68-5) and H2O2 (CAS. No: 7722-84-1) were obtained from Applichem. Lymphocyte Separation Media (CAS No: JO100–840) was acquired from Cegrogen.
Selection of concentrations for use in the study
To determine the concentrations to be used in this study, LD50 doses of Carmoisine (Rat oral: 10 mg/kg and mouse oral 8 mg/kg) and Ponceau 4R (mouse oral 8 mg/kg) obtained from literature searches were taken as reference [29]. For this purpose, Carmoisine and Ponceau 4R at concentrations of 4.69, 9.38, 18.75, 37.5, 75, 150, and 300 μg/mL were tested in a preliminary experiment. Mitotic index (MI) values obtained from concentrations of 4.69, 9.38, and 18.75 μg/mL were not different from those of 37.50 μg/mL. The value that decreased the MI by approximately 50% was chosen as the highest concentration. Depending on the mitotic index result, 37.5, 75, 150, and 300 μg/mL were selected as experimental concentrations.
Chromosomal aberrations and sister chromatid exchange assay
The chromosomal abnormality test was performed according to the method of Evans30 with some modifications.31 The sister chromatid exchange test was performed using the method of Perry and Wolff, 197432 with some modifications.33
Heparinized peripheral blood (0.2 mL) obtained from donors was added to sterile 2.5 mL tubes containing LymphoPlus Medium. Then, 10 μg/mL of BrdU (5-Bromo-2- deoxyuridine = Bromodeoxyuridine) solution was added to the same tubes. The tubes were incubated at 37 °C for 72 h. Four different concentrations (37.5, 75, 150, 300 μg/mL) of Carmoisine and Ponceau 4R were added to the medium at 24 and 48 h from the beginning of the culture. At the same time, distilled water was added as the negative control, and Mitomycin C was added as the positive control (0.20 μg/mL). Colchicine at 0.06 μg/mL was added to all tubes 2 h before the end of the culture (70th h of culture). At the end of 72 h of culture time, the tubes were removed from the oven, centrifuged at 1200 rpm for 10 min, and the supernatant was discarded. 0.075 M KCl (potassium chloride) solution was added to the remaining part. The culture tubes were incubated with this solution at 37 °C for 20 min and centrifuged again. Afterward, the supernatant was discarded, and a cold 3:1 methanol: acetic acid mixture was added to the cells and kept at +4 °C for 45 min. The fixation process was repeated two more times in the same way. Then, the cell suspension at the bottom of the tube was homogenized by the pipetting method and dripped onto the cold slides so they did not fall on each other.
The slides were dried at room temperature for 24 h and subjected to fluorescent + Giemsa staining to observe SCEs.33 To determine the replication index (RI), 100 cells from each treatment group (a total of 200) were analyzed. The following formula was used to calculate the replication index: RI = [(1xM1) + (2xM2) + (3xM3)]/N (N: number of cells observed, M1, M2, and M3: number of cells undergoing first, second and third divisions).34
Cytokinesis block micronucleus cytome assay
The cytokinesis block micronucleus cytome test was performed using the method of Fenech, 200735 with some modifications applied by Yuzbasioglu and Avuloglu-Yilmaz.36 Accordingly, 0.2 mL of heparinized peripheral blood from each donor was inoculated into tubes containing 2.5 mL of LymphoPlus Medium.
The culture tubes were incubated at 37 °C for 72 h. Four different concentrations of Carmoisine and Ponceau 4R (37.5, 75, 150, 300 μg/mL) were added to the medium 24 h after the start of the culture. In addition, a positive control (Mitomycin C, 0.20 μg/mL) and a negative control (distilled water) were also used. At 44 h of incubation, cytokinesis was inhibited by adding cytochalasin-B (5.2 μg/mL) to the culture medium. The cells were centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. 0.075 M KCI hypotonic solution was added to the tubes. Fixation was then performed with a cold 3:1 mixture of methanol and acetic acid, and the tubes were kept at +4 °C for 15 min. This process was repeated three times, and formaldehyde (1 mL of 40% formaldehyde was added to a 40 mL volume of fixative) was added to the final fixative. After centrifugation, the suspension was spread dropwise onto clean slides, and these slides were dried at room temperature for 24 h.
A total of 2,000 binucleated cells (1,000 from each donor) were scored to determine the frequency of MN, NPBs, and NBUDs, while a total of 1,000 cells (500 each from two female donors) were evaluated to determine the proliferation index (PI). The frequency of micronuclei per cell was determined using the formula [1x (1MN) +2x (2MN) + 3x (3MN + 4MN)]/N (N is the total number of cells examined) and the proliferation index was determined using the formula [1x (M1) +2x(M2) + 3x(M3) + 4x(M4)]/n (n is the total number of cells examined).
Comet assay
Single-cell gel electrophoresis (SCGE) or Comet technique was performed by applying the method of Singh et al. (1988)37 and some modifications used by Avuloğlu-Yılmaz et al. (2017).38 Cell viability was found to be >97% using the Trypan Blue Exclusion Test in lymphocytes isolated from peripheral blood obtained from donors using Lymphocyte Separation Medium. Isolated human lymphocytes were incubated with increasing concentrations of Carmoisine and Ponceau 4R food dyes for 1 h at 37 °C. Afterward, they were spread on agar-coated slides. The slides were placed in lysis solution and kept in alkaline electrophoresis buffer, and then electrophoresis (25 V, 300 mA) was performed. The slides in a neutralization buffer were then stained with EtBr and examined. Two hundred cells, 100 from each donor for each concentration, were examined under a fluorescence microscope using the Comet assay program (Comet Assay IV, Perceptive Instruments Ltd, UK).
Statistical analysis
Regression analysis was applied using IBM SPSS 23.0 computer program to reveal the concentration-effect relationship for mitotic index, replication index, SCE/cell number, MN/cell, nuclear bud and nucleoplasmic bridge frequency, abnormal cell frequency, CA/cell number, % tail intensity, tail length and tail moment.
Mitotic index, replication index, the frequency of abnormal cells, CA/Cell, the frequency of micronucleus per cell, the frequency of nuclear bud and nucleoplasmic bridge, and proliferation index obtained from treatment and control groups were analyzed by z test, sister chromatid exchange, and comet test results were analyzed by One-way ANOVA (Tukey test P < 0.05) test.
Results
Carmoisine and Ponceau 4R increased the percentage of abnormal cells and the frequency of chromosomal abnormalities per cell at both treatment periods. However, this increase was significant only at the highest concentration (300 μg/mL) for both parameters (Tables 1 and 2). Five types of structural abnormalities were observed in both treatment periods. Chromatid and chromosome breaks, sister chromatid union, fragment, chromatid exchange, and dicentric chromosomes were observed in Carmoisine and Ponceau 4R treatments. In addition, polyploidy, a numerical abnormality, was also observed less frequently (Tables 1 and 2).
Table 1.
Effect of Carmoisine on CAs in human peripheral lymphocytes.
| Test Substance | Treatment | Aberrations | Abnormal cell ± SE (%) | CA/cell ± SE | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| h | Conc (μg/mL) | ctb | csb | f | scu | dic | ce | p | |||
| Control | 24 | 0.00 | 2 | – | 1 | – | – | – | – | 1.50 ± 0.86 | 0.015 ± 0.008 |
| MMC | 24 | 0.20 | 9 | 2 | 1 | 2 | – | 1 | 2 | 8.50 ± 1.97 | 0.080 ± 0.019 |
| Carmoisine | 24 | 37.5 | 3 | – | – | 1 | – | – | 1 | 2.50 ± 1.10 | 0.025 ± 0.011 |
| 75 | 4 | – | 1 | – | – | – | – | 2.50 ± 1.10 | 0.025 ± 0.011 | ||
| 150 | 4 | 1 | – | 1 | – | – | 1 | 3.50 ± 1.29 | 0.035 ± 0.012 | ||
| 300 | 6 | 1 | 1 | 2 | – | – | – | 5.00 ± 1.54a | 0.050 ± 0.015a | ||
| Control | 48 | 0.00 | 2 | – | 1 | – | – | – | – | 1.50 ± 0.86 | 0.015 ± 0.008 |
| MMC | 48 | 0.20 | 10 | 3 | 1 | 2 | 1 | 1 | 1 | 9.50 ± 2.07 | 0.090 ± 0.020 |
| Carmoisine | 48 | 37.5 | 2 | – | 1 | 1 | – | – | – | 2.00 ± 0.98 | 0.020 ± 0.009 |
| 75 | 3 | 1 | – | 2 | – | – | – | 2.50 ± 1.10 | 0.020 ± 0.009 | ||
| 150 | 4 | 1 | 1 | 1 | – | 1 | 1 | 4.50 ± 1.46 | 0.045 ± 0.014 | ||
| 300 | 8 | 2 | 1 | 1 | – | – | 1 | 6.50 ± 1.74a | 0.060 ± 0.016a | ||
| Frequency of abnormalities | 57.63 | 10.17 | 8.48 | 15.25 | 0.00 | 1.70 | 6.77 | ||||
h: hour, Conc: Concentration,s ctb: Chromatid break, csb: chromosome break, scu: sister chromatid union, dic: dicentric chromosome, ce: chromatid exchange, f: fragment, p: polyploidy, SE: Standart Error
aSignificantly different from the control P < 0.05 (z test).
Table 2.
Effect of Ponceau 4R on CAs in human peripheral lymphocytes.
| Test Substance | Treatment | Aberrations | Abnormal cell ± SE (%) | CA/cell ± SE | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| h | Conc (μg/mL) | ctb | csb | f | scu | dic | ce | p | r | |||
| Control | 24 | 0.00 | 1 | – | – | 1 | – | – | 2 | – | 2.00 ± 0.98 | 0.020 ± 0.019 |
| MMC | 24 | 0.20 | 2 | – | 3 | 6 | – | – | 2 | – | 6.50 ± 1.74 | 0.060 ± 0.016 |
| Ponceau 4R | 24 | 37.5 | 2 | – | 3 | 1 | – | – | 1 | – | 3.50 ± 1.29 | 0.035 ± 0.012 |
| 75 | 1 | – | 2 | 4 | – | – | 1 | – | 4.00 ± 1.38 | 0.030 ± 0.012 | ||
| 150 | 2 | – | 3 | 1 | – | – | 1 | – | 3.50 ± 1.29 | 0.035 ± 0.012 | ||
| 300 | 1 | 1 | 6 | 5 | – | – | 2 | – | 7.50 ± 1.86a | 0.065 ± 0.017b | ||
| Control | 48 | 0.00 | 2 | – | – | 2 | – | – | 1 | – | 2.50 ± 1.10 | 0.025 ± 0.011 |
| MMC | 48 | 0.20 | 10 | 1 | 4 | 7 | – | 1 | 3 | 1 | 13.5 ± 2.41 | 0.130 ± 0.023 |
| Ponceau 4R | 48 | 37.5 | 2 | – | 4 | 3 | – | – | 2 | – | 5.50 ± 1.61 | 0.055 ± 0.016 |
| 75 | 1 | – | 2 | 5 | – | – | 1 | – | 4.50 ± 1.46 | 0.045 ± 0.014 | ||
| 150 | 4 | – | 1 | 6 | 1 | – | – | – | 6.00 ± 1.67 | 0.060 ± 0.016 | ||
| 300 | 3 | 1 | 1 | 9 | 1 | – | 1 | – | 8.00 ± 1.91b | 0.080 ± 0.019b | ||
| Frequency of abnormalities | 18.82 | 2.35 | 25.89 | 40.00 | 2.35 | 0.00 | 10.59 | 0.00 | ||||
h: hour, Conc: Concentrations, ctb: Chromatid break, csb: chromosome break, scu: sister chromatid union, dic: dicentric chromosome, ce: chromatid exchange, f: fragment, p: polyploidy, r:ring, SE: Standart Error
aSignificantly different from the control P < 0.01 (z test)
bSignificantly different from the control P < 0.05 (z test).
Carmoisine and Ponceau 4R significantly increased the frequency of sister chromatid exchange in 24- and 48-h treatments, only at the highest concentration (300 μg/mL) compared to the control (except Carmoisine, at 48 h) (Tables 3 and 4).
Table 3.
Effect of Carmoisine on SCE, and RI in human peripheral lymphocytes.
| Test Substance | Treatment | Min-max SCE | SCE/cell ±SE | M1 | M2 | M3 | RI ± SE | |
|---|---|---|---|---|---|---|---|---|
| Hour | Concentrations (μg /mL) | |||||||
| Control | 24 | 0.00 | 0–11 | 3.06 ± 0.33 | 68 | 56 | 74 | 2.01 ± 0.05 |
| MMC | 24 | 0.20 | 4–22 | 11.4 ± 0.64 | 61 | 57 | 82 | 2.10 ± 0.05 |
| Carmoisine | 24 | 37.5 | 0–9 | 3.44 ± 0.31 | 71 | 55 | 74 | 2.01 ± 0.06 |
| 75 | 0–10 | 4.30 ± 0.36 | 66 | 56 | 81 | 2.10 ± 0.06 | ||
| 150 | 0–10 | 3.72 ± 0.35 | 61 | 56 | 85 | 2.14 ± 0.05 | ||
| 300 | 0–11 | 4.38 ± 0.38+ | 55 | 57 | 88 | 2.16 ± 0.05 | ||
| Control | 48 | 0.00 | 3–14 | 3.22 ± 0.38 | 68 | 59 | 73 | 2.02 ± 0.05 |
| MMC | 48 | 0.20 | 2–29 | 9.20 ± 1.09 | 80 | 53 | 67 | 1.93 ± 0.06 |
| Carmoisine | 48 | 37.5 | 0–4 | 3.24 ± 0.25 | 72 | 55 | 73 | 2.00 ± 0.06 |
| 75 | 1–11 | 3.74 ± 0.27 | 72 | 53 | 75 | 2.01 ± 0.06 | ||
| 150 | 0–8 | 3.68 ± 0.27 | 68 | 53 | 79 | 2.05 ± 0.06 | ||
| 300 | 0–12 | 3.74 ± 0.41 | 70 | 58 | 72 | 2.01 ± 0.05 | ||
+Significantly different from the control P < 0.05 (One Way ANOVA-Tukey test).
Table 4.
Effect of Ponceau 4R on SCE and RI in human peripheral lymphocytes.
| Test Substance | Treatment | Min-max SCE | SCE/cell ±SE | M1 | M2 | M3 | RI ± SE | |
|---|---|---|---|---|---|---|---|---|
| Hour | Concentrations (μg /mL) | |||||||
| Control | 24 | 0.00 | 0–11 | 3.32 ± 0.35 | 51 | 66 | 83 | 2.16 ± 0.05 |
| MMC | 24 | 0.20 | 4–22 | 8.26 ± 0.55 | 61 | 67 | 72 | 2.05 ± 0.06 |
| Ponceau 4R | 24 | 37.5 | 0–9 | 3.96 ± 0.34 | 56 | 60 | 84 | 2.14 ± 0.05 |
| 75 | 0–10 | 4.46 ± 0.34 | 62 | 57 | 81 | 2.09 ± 0.06 | ||
| 150 | 0–10 | 4.72 ± 0.35 | 57 | 62 | 81 | 2.12 ± 0.05 | ||
| 300 | 0–11 | 6.04 ± 0.41+ | 54 | 61 | 85 | 2.15 ± 0.05 | ||
| Control | 48 | 0.00 | 3–14 | 2.94 ± 0.29 | 56 | 65 | 79 | 2.11 ± 0.05 |
| MMC | 48 | 0.20 | 2–29 | 10.24 ± 0.75 | 62 | 67 | 70 | 2.03 ± 0.05 |
| Ponceau 4R | 48 | 37.5 | 0–4 | 4.06 ± 0.34 | 51 | 60 | 89 | 2.19 ± 0.05 |
| 75 | 1–11 | 4.12 ± 0.32 | 59 | 58 | 83 | 2.12 ± 0.04 | ||
| 150 | 0–8 | 4.24 ± 0.36 | 53 | 61 | 86 | 2.16 ± 0.05 | ||
| 300 | 0–12 | 6.28 ± 0.36+ | 55 | 62 | 83 | 2.14 ± 0.05 | ||
+Significantly different from the control P < 0.05 (One Way ANOVA-Tukey test).
The mitotic index decreased significantly at almost all concentrations (except the lowest one) in the 24-h treatment with Carmoisine and Ponceau 4R (Figs. 2 and 3). In addition, MI showed a significant decrease at the two highest concentrations of Carmoisine and all concentrations of Ponceau 4R in the 48 h treatments compared to the control (Figs. 2 and 3). Regression analyses revealed that there was a strong and opposite correlation between the concentrations and MI (r = −0.76 at 24 h, and r = −0.89 at 48 h for Carmoisine, and r = −0.76 at 24 h, and r = −0.89 at 48 h for Ponceau 4R).
Figure 2.
Mitotic index values of 24 and 48-h treatment of Carmoisine.
Figure 3.
Mitotic index values of 24 and 48 h treatment of Ponceau 4R.
Carmoisine did not generate a significant change in the frequencies of MN and NPBs. However, it significantly increased the frequency of NBUDs at the highest concentration (300 μg/mL) compared to the control. On the other hand, Ponceau 4R did not induce a significant change in the frequency of MN, NPBs, and NBUDs (Figs. 4 and 5).
Figure 4.
Effect of Carmoisine on micronucleus, nuclear bud, and nucleoplasmic bridge frequencies in human lymphocytes.
Figure 5.
Effect of Ponceau 4R on micronucleus, nuclear bud, and nucleoplasmic bridge frequencies in human lymphocytes.
None of the food colorings significantly affected either the proliferation index (PI) or the replication index (RI) compared to controls (Tables 3–5).
Table 5.
Effect of Carmoisine and Ponceau 4R on PI in human lymphocytes.
| Test Substance | Treatment | Proliferation Index (PI) ± SE | |
|---|---|---|---|
| Hour | Concentrations (μg /mL) | ||
| Control | 48 | 0.00 | 1,66 ± 0,40 |
| MMC | 48 | 0.20 | 1,61 ± 0,39 |
| Carmoisine | 48 | 37.5 | 1,67 ± 0,40 |
| 75 | 1,58 ± 0,39 | ||
| 150 | 1,35 ± 0,36 | ||
| 300 | 1,47 ± 0,38 | ||
| Control | 48 | 0.00 | 1,75 ± 0,41 |
| MMC | 48 | 0.20 | 2,14 ± 0,45 |
| Ponceau 4R | 48 | 37.5 | 1,63 ± 0,20 |
| 75 | 1,55 ± 0,39 | ||
| 150 | 1,56 ± 0,39 | ||
| 300 | 1,60 ± 0,39 | ||
Carmoisine and Ponceau 4R did not significantly affect the comet tail intensity at any concentration compared to the controls (Fig. 6). However, these two food dyes significantly increased comet tail length at higher concentrations than controls (Carmoisine at 300 μg/mL and Ponceau 4R at 150 and 300 μg/mL) (Fig. 7). Ponceau 4R significantly increased tail moment compared to controls only at 300 μg/mL concentration. Carmoisine did not induce any change in tail moment compared to the control (Fig. 8).
Figure 6.
Effect of Carmoisine and Ponceau 4R on comet tail intensity.
Figure 7.
Effect of Carmoisine and Ponceau 4R on comet tail length.
Figure 8.
Effect of Carmoisine and Ponceau 4R on comet tail moment.
Discussion
Approximately 65% of azo dyes are used as food additives, mainly in soft drinks, jam, confectionery, pickles, and so on.39,40 When azo food dyes are ingested orally, they are thought to exert their toxic effects by reduction of the dyes by intestinal microflora, i.e. cleavage of azo bonds leading to the formation of aromatic amines (which can then be N-hydroxylated or N-acetylated).40,41 Therefore, there is a concern that azo food colorings may pose health risks.40,42 Carmoisine and Ponceau 4R, two azo food dyes that impart a red color, are added to foods such as commonly consumed beverages, confectionery, and pickles.8,10
Carmoisine can cause attention disorders and is also reported to have neurological and allergenic potential.43 It has been reported that the aromatic ring structure of Ponceau 4R and its azo functional group may harm the human body and cause genetic defects and cytotoxicity. Studies have stated that aqueous solutions of these food dyes are mixed with waste environmental waters and thus may cause ecological problems.44–46
In addition to the concern that food additives may negatively affect human health, it is also essential to determine the damage they may cause to genetic material. Due to this worry, they are currently evaluated by short-term genotoxicity tests to assess their genotoxic potential. Genotoxicity tests enable us to understand the damage mechanism of various physical and chemical agents on genetic material.47 As a result of genotoxicity tests, it is possible to evaluate the chemical risks of the products consumed. This information is essential for consumers. Because the adverse health effects generated by genotoxic effects are irreversible and can have serious consequences.48 Among genotoxicity tests, chromosomal abnormality, sister chromatid exchange, cytokinesis block micronucleus cytome, and comet tests are frequently applied in human peripheral lymphocytes. All these methods, except the SCE test, are those recommended by the OECD for testing chemicals. Although the SCE test was excluded from the OECD guidelines in 2014, it is a test often used to support the results of other genotoxicity tests.
In this study, the genotoxic effects of food dyes Carmoisine (E-122) and Ponceau 4R (E-124) were investigated by four different genotoxicity tests (chromosome abnormalities, sister chromatid exchange, cytokinesis block micronucleus cytome and comet) under in vitro conditions. Mitotic index, replication index, and proliferation index values were also determined with these tests. When the results of all genotoxicity tests are evaluated together, it is seen that these two food dyes exhibit genotoxic potential only at high concentrations. Carmoisine and Ponceau 4R are thought to have cytotoxic potential since they cause decreases in MI. However, they have no significant effect on the replication and proliferation indexes.
There are limited studies investigating the genotoxic effects of Carmoisine and Ponceau 4R colorants. In a 2011 study, the genotoxicity of Carmoisine and Ponceau 4R was analyzed using a micronucleus test. Concentrations of 100, 200, and 300 μg/mL of food dyes were applied to human peripheral blood cells, and a significant increase in MN frequency was observed compared to the control.28 In our study, the CBMN-Cyt test was applied, and no change in MN frequency was observed, while a significant increase in the frequency of nuclear bud was found. While there are no other studies in the literature on the genotoxicity of Carmoisine and Ponceau 4R in human peripheral lymphocytes, there are studies in which genotoxic effects were examined with different test systems.
According to the EFSA Panel 200949 and 201350 report, it was concluded that Carmoisine has no concern about genotoxicity, as most studies showed no genotoxic effect. No mutagenic effect was found when E. coli was tested for cytotoxicity and mutagenicity with S. typhimurium (strains TA1535, TA1538, TA100, and TA98).18 No evidence of mutagenic potential was obtained after exposure of S. typhimurium (TA1538) to Carmoisine with or without metabolic activation.19 Without microsomal activation, Carmoisine did not induce mitotic gene conversion in Saccharomyces cerevisiae (BZ 34).51 In an in vivo study in mice, it did not increase chromosomal damage.20 Several in vitro studies have been carried out, including the Salmonella/microsome test, the mouse lymphoma TK +/− test, the Chinese hamster ovary cell chromosome abnormality and sister chromatid exchange test, and the rat hepatocyte/DNA repair test, but no further details were provided.21,52,53 A chromosomal abnormality was analyzed in another study, and the possibility of inducing mitotic index and chromosomal modifications was explained.54 Raya et al. performed an alkali comet test in albino rats to evaluate the safety of various food additive mixtures, including Carmoisine. Food additives were given orally to rats divided into groups for thirty days. Their effects on many parameters were examined, such as hemoglobin, renal function, AST, ALT and ALP activities, total protein, and albumin levels. The results showed that all combinations of food additives significantly decreased hemoglobin concentration compared to the control group. As the number of food additives increased, diverse degrees of genotoxic effects grew in the brain, kidney, and liver.55 In 2019, in a study examining the toxicity and carcinogenicity of Carmoisine, this azo dye was administered orally to mice at low, medium, and high levels (4, 200, and 400 mg/kg/body weight, respectively) for 120 days. RT-PCR results showed that Carmoisine significantly increased the expression of the Bcl-x and PARP genes and decreased the expression of the p53 gene in mouse liver tissues. This study revealed that high doses (400 mg/kg) of Carmoisine caused renal failure and hepatotoxicity. It was also explained that it may affect liver oncogenesis.56
Diverse genotoxicity studies of Ponceau 4R were included in the EFSA 2009 and 2013 panels. In E. coli strains, Ponceau 4R was reported to have no mutagenic potential (Lück and Rickerl, 1960). Clastogenic activity and chromosome aberrations were recorded in bone marrow cells of male mice at a minimum effective intraperitoneal dose.25 S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 with and without S9 were negative in the Ames and mouse lymphoma TK+/− tests. S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94, and TA98 were also examined by the Ames test and chromosomal abnormality test in hamster fibroblasts. Ponceau 4R was reported to be negative in the Ames test and positive in the chromosomal abnormality test.24 A micronucleus test was performed on mice, and it was observed that Ponceau 4R was negatively influential.27 It was also found to cause significant increases in DNA damage in the comet test in mice.26 Genotoxic effects of Ponceau 4R were investigated by in vivo micronucleus test. Groups of Swiss albino mice were treated with doses of 0.5, 1.0, and 2 g/kg, and the effects were determined at 24 and 48 h. Bone marrow polychromatic (PCE) and normochromatic erythrocytes (NCE) and PCE with micronuclei (MNPCE) were analyzed, and Ponceau 4R showed dose- and time-dependent genotoxicity and time- and sex-dependent systemic toxicity. The authors explained that it may produce clastogenic and aneugenic effects.7 In another study, it was determined by comet assay that Ponceau 4R induced DNA lesions in the stomach, colon, bladder, liver, kidney, and lung cells of male ddY mice. However, it was not genotoxic for bone marrow and brain cells.57
Carmoisine and Ponceau 4R metabolism produces sulfonated aromatic amines. Jung et al. (1992)58 studied the genotoxicity of a series of sulfonated aromatic amines. The genotoxicity of sulfonated aromatic amines was compared with their non-sulfonated analogs to evaluate the effect of sulfonating on the genotoxic potential of phenylamines and naphthylamines. In general, sulfonated phenylamines and naphthylamines were non-mutagenic against Salmonella in Ames tests. Other in vitro and in vivo test systems for some sulfonated aromatic amines showed no genotoxicity. Based on the available data, it is concluded that sulfonated aromatic amines have no or very low genotoxic potential, in contrast to their non-sulfonated analogs. The study, therefore, showed that exposure to sulfonated aromatic amines derived by metabolic breakdown or present as contaminants in coloring does not pose any significant genotoxic risk.49 However, contrary to this, some studies show that their metabolism generates the toxic effects of commonly used azo dyes. Enzyme-mediated azo reduction takes place, resulting in the formation of active aromatic amines that attack DNA. Consumption of these substances in amounts higher than the recommended daily intake (ADI) can cause adverse effects in mammals.59
This study observed that the genotoxic potential of both azo dyes was similar and induced genotoxic effects, especially at high concentrations, while low concentrations did not. Some of the studies conducted in the past explain that these food colors carry genotoxic risks, while others emphasize that they do not have such an effect. In this study, while only high concentrations showed genotoxic effects in human lymphocytes, stressing the importance of concentration in these contradictory results, the results were obtained without metabolic activation.
In conclusion, in this study, the genotoxicity of Carmoisine and Ponceau 4R on human lymphocytes was examined by four different complementary genotoxicity tests. As a result, both Carmoisine and Ponceau 4R demonstrated genotoxic potential at high concentrations in human lymphocytes. These findings emphasize the need for further investigation into the long-term health risks associated with the consumption of these food dyes. Therefore, using low doses of these chemicals in products such as food and medicine that people are frequently exposed to is also essential to investigate. On the other hand, it should be remembered that these colorants are not used alone in products; they are present with many food additives. Therefore, it should be taken into consideration that a cocktail effect may occur. In particular, the findings of this study were obtained under in vitro conditions and need to be supported using metabolic activation and/or in vivo studies. However, adverse health effects can be minimized if the dose used is strictly controlled, considering the in vitro results.
Contributor Information
Sadriye Gokce Kara, Gazi University, Science Faculty, Department of Biology, Genetic Toxicology Laboratory, Ankara, 06170, Turkey.
Deniz Yuzbasioglu, Gazi University, Science Faculty, Department of Biology, Genetic Toxicology Laboratory, Ankara, 06170, Turkey.
Ece Avuloglu-Yilmaz, Amasya University, School of Technical Sciences, Department of Health Information Systems, Amasya, 05000, Turkey.
Fatma Unal, Gazi University, Science Faculty, Department of Biology, Genetic Toxicology Laboratory, Ankara, 06170, Turkey.
Author contributions
All authors contributed to the study conception and design. Investigation, Formal Analysis, Visualization were performed by Sadriye Gokce Kara and Ece Avuloglu-Yilmaz. Supervision, Resources, Writing–Original Draft Preparation were performed by Deniz Yuzbasioglu. Writing - Review & Editing, Visualization, Conceptualization were performed by Deniz Yuzbasioglu and Fatma Unal. All authors have commented on the manuscript, tread and approved the final manuscript.
Funding
None declared.
Conflicts of interest: The Authors declare that there is no conflict of interest.
Data availability statement
The analyzed data sets generated during the present study are available from the corresponding author upon reasonable request.
References
- 1. Chung KT. Azo dyes and human health: a review. J Environ Sci Health Part C. 2016:34:233–261. 10.1080/10590501.2016.1236602 [DOI] [PubMed] [Google Scholar]
- 2. Ramos-Souza C, Bandoni DH, Bragotto APA, de Rosso VV. Risk assessment of azo dyes as food additives: revision and discussion of data gaps toward their improvement. Compr Rev Food Sci Food Saf. 2023:22:380–407. 10.1111/1541-4337.13072 [DOI] [PubMed] [Google Scholar]
- 3. Batada A, Jacobson MF. Prevalence of artificial food colors in grocery store products marketed to children. Clin Pediatr. 2016:55:1113–1119. 10.1177/0009922816651621 [DOI] [PubMed] [Google Scholar]
- 4. Sweeney EA, Chipman JK, Forsythe SJ. Evidence for direct-acting oxidative genotoxicity by reduction products of azo dyes. Environ Health Perspect. 1994:102 Suppl 6:119–122. 10.1289/ehp.94102s6119 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Nachiyar V, Rajakumar S. Mechanism of navitan fast blue S5R degradation by Pseudomonas aeruginosa. Chemosphere. 2004:57:165–169. 10.1016/j.chemosphere.2004.05.030 [DOI] [PubMed] [Google Scholar]
- 6. Novotný C et al. Comparative use of bacterial, algal and protozoan tests to study toxicity of azo- and anthraquinone dyes. Chemosphere. 2006:63:1436–1442. 10.1016/j.chemosphere.2005.10.002 [DOI] [PubMed] [Google Scholar]
- 7. de Souza CSH et al. Genotoxic and cytotoxic potential of food azodyes: preclinical safety assessment using the in vivo micronucleus assay. Braz J Dev. 2022:8:57227–57247. 10.34117/bjdv8n8-158 [DOI] [Google Scholar]
- 8. European Food Safety Authority (EFSA) . Refined exposure assessment for Azorubine/Carmoisine (E 122). EFSA J. 2015a:13:4072. 10.2903/j.efsa.2015.4072 [DOI] [Google Scholar]
- 9. Pereira H et al. Painting the picture of food colouring agents: near-ubiquitous molecules of everyday life–a review. Trends Food Sci. 2023:143:104249. 10.1016/j.tifs.2023.104249 [DOI] [Google Scholar]
- 10. European Food Safety Authority (EFSA) . Refined exposure assessment for Ponceau 4R (E 124). EFSA J. 2015b:13:4073. 10.2903/j.efsa.2015.4073 [DOI] [Google Scholar]
- 11. Zeence M. Food colorants: introduction to the chemistry of food. Vol. 313. London, UK: Academic Press, Elsevier; 2020. p. 344. 10.1016/B978-0-12-809434-1.00008-6 [DOI] [Google Scholar]
- 12. Stratton MR, Campbell PJ, Futreal PA. The cancer genome. Nature. 2009:458:719–724. 10.1038/nature07943 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Bonassi S, El-Zein R, Bolognesi C, Fenech M. Micronuclei frequency in peripheral blood lymphocytes and cancer risk: evidence from human studies. Mutagenesis. 2011:26:93–100. 10.1093/mutage/geq075 [DOI] [PubMed] [Google Scholar]
- 14. Roussel C, Witt KL, Shaw PB, Connor TH. Meta-analysis of chromosomal aberrations as a biomarker of exposure in healthcare workers occupationally exposed to antineoplastic drugs. Mutat Res/Rev Mutat Res. 2019:781:207–217. 10.1016/j.mrrev.2017.08.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Avuloglu Yilmaz E, Yuzbasioglu D, Unal F. Investigation of genotoxic effect of octyl gallate used as an antioxidant food additive in in vitro test systems. Mutagenesis. 2023:38:151–159. 10.1093/mutage/gead005 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Lück H, Rickerl E. Lebensmittelzusatzstoffe und mutagene wirkung. Vi mitteilung. prüfung der in westdeutschland zugelassenen und ursprünglich vorgeschlagenen lebensmittelfarbstoffe auf mutagene wirkung an E. coli Z Lebensm-Unters Forsch. 1960:112:157–174. 10.1007/BF01454513 [DOI] [Google Scholar]
- 17. Garner RC, Nutman CA. Testing of some azo dyes and their reduction products for mutagenicity using salmonella typhimurium TA 1538. Mutat Res. 1977:44:9–19. 10.1016/0027-5107(77)90110-5 [DOI] [PubMed] [Google Scholar]
- 18. Viola M, Nosotti A. Applicazione del test di Ames su alcuni coloranti. Boll Chim Farm. 1978:117:402–415. [PubMed] [Google Scholar]
- 19. Haveland-Smith RB, Combes RD. Screening of food dyes for genotoxic activity. Food Cosmet Toxicol. 1980:18:215–221. 10.1016/0015-6264(80)90097-8 [DOI] [PubMed] [Google Scholar]
- 20. Durnev AD, Oreshchenko AV, Kulakova AV, et al. Analysis of cytogenetic activity of food dyes. Vop Med Khim. 1995:41:50–53 [PubMed] [Google Scholar]
- 21. Gulati DK, Witt K, Anderson B, Zeiger E, Shelby MD. Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. III. Results with 27 chemicals. Environ Mol Mutagen. 1989:13:133–193. 10.1002/em.2850130208 [DOI] [PubMed] [Google Scholar]
- 22. Longstaff E, McGregor DB, Harris WJ, Robertson JA, Poole A. A comparison of the predictive values of the salmonella/microsome mutation and BHK21 cell transformation assays in relation to dyestuffs and similar materials. Dyes Pigments. 1984:5:65–82. 10.1016/0143-7208(84)80006-6 [DOI] [Google Scholar]
- 23. Cameron TP, Hughes TJ, Kirby PE, Fung VA, Dunkel VC. Mutagenic activity of 27 dyes and related chemicals in the salmonella/microsome and mouse lymphoma TK+/− assays. Mutat Res. 1987:189:223–261. 10.1016/0165-1218(87)90056-5 [DOI] [PubMed] [Google Scholar]
- 24. Ishidate MJ et al. Primary mutagenicity screening of food additives currently used in Japan. Food Chem Toxicol. 1984:22:623–636. 10.1016/0278-6915(84)90271-0 [DOI] [PubMed] [Google Scholar]
- 25. Agarwal K, Mukherjee A, Sharma A. In vivo cytogenetic studies on male mice exposed to Ponceau 4R and beta-carotene. Cytobios. 1993:74:23–28. [PubMed] [Google Scholar]
- 26. Tsuda S et al. DNA damage induced by red food dyes orally administered to pregnant and male mice. Toxicol Sci. 2001:61:92–99. 10.1093/toxsci/61.1.92 [DOI] [PubMed] [Google Scholar]
- 27. Hayashi M, Kishi M, Sofuni T, Ishidate M. Micronucleus test in mice on 39 food additives and eight miscellaneous chemicals. Food Chem Toxicol. 1988:26:487–500. 10.1016/0278-6915(88)90001-4 [DOI] [PubMed] [Google Scholar]
- 28. Swaroop VR, Roy DD, Vijayakumar T. Genotoxicity of synthetic food colorants. J Food Sci Eng. 2011:1:128–134. [Google Scholar]
- 29. Gaunt IF, Farmer M, Grasso P, Gangolli SD. Acute (mouse and rat) and short-term (rat) toxicity studies on Ponceau 4R. Food Cosmet Toxicol. 1967:5:187–194. 10.1016/S0015-6264(67)82966-3 [DOI] [PubMed] [Google Scholar]
- 30. Evans HJ. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. In: Kilbey BJ, Legator M, Nichols W, Ramel C, editors. Handbook of mutagenicity test procedures. New York, NY, USA: Elsevier Science Publishing; 1984. pp. 405–424. 10.1016/B978-0-444-80519-5.50023-7 [DOI] [Google Scholar]
- 31. Yuzbasioglu D, Çelik M, Yılmaz S, Unal F, Aksoy H. Clastogenicity of the fungicide afugan in cultured human lymphocytes. Mutat Res Genet Toxicol Environ Mutagen. 2006:604:53–59. 10.1016/j.mrgentox.2006.01.001 [DOI] [PubMed] [Google Scholar]
- 32. Perry P, Wolff S. New giemsa method for the differential staining of sister chromatids. Nature. 1974:251:156–158. 10.1038/251156a0 [DOI] [PubMed] [Google Scholar]
- 33. Speit G, Haupter S. On the mechanism of differential giemsa staining of bromodeoxyuridine-substituted chromosomes. Hum Genet. 1985:70:126–129. 10.1007/BF00273070 [DOI] [PubMed] [Google Scholar]
- 34. Schneider EL, Nakanishi Y, Lewis J, Sternberg H. Simultaneous examination of sister chromatid exchanges and cell replication kinetics in tumor and normal cells in vivo. Cancer Res. 1981:41:4973–4975. [PubMed] [Google Scholar]
- 35. Fenech M. Cytokinesis-block micronucleus cytome assay. Nat Protoc. 2007:2:1084–1104. 10.1038/nprot.2007.77 [DOI] [PubMed] [Google Scholar]
- 36. Yuzbasioglu D, Avuloglu-Yilmaz E. Mikronukleus testi. In: Unal F, Yuzbasioglu D, editors. Genetik Toksikoloji. Ankara, Türkiye: Nobel Yayıncılık; 2022. pp. 219–249. [Google Scholar]
- 37. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988:175:184–191. 10.1016/0014-4827(88)90265-0 [DOI] [PubMed] [Google Scholar]
- 38. Avuloglu-Yilmaz E, Yuzbasioglu D, Ozcelik AB, Ersan S, Ünal F. Evaluation of genotoxic effects of 3-methyl-5-(4-carboxycyclohexylmethyl)-tetrahydro-2H-1, 3, 5-thiadiazine-2-thione on human peripheral lymphocytes. Pharm Biol. 2017:55:1228–1233. 10.1080/13880209.2017.1296000 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39. Sun R et al. Simple and sensitive electrochemical detection of sunset yellow and Sudan I in food based on AuNPs/Zr-MOF-graphene. Food Control. 2023:145:1–8. 10.1016/j.foodcont.2022.109491 [DOI] [Google Scholar]
- 40. Barciela P, Perez-Vazquez A, Prieto MA. Azo dyes in the food industry: features, classification, toxicity, alternatives, and regulation. Food and Chem Toxicol. 2023:178:113935. 10.1016/j.fct.2023.113935 [DOI] [PubMed] [Google Scholar]
- 41. Kaya SI, Cetinkaya A, Ozkan SA. Latest advances on the nanomaterials-based electrochemical analysis of azo toxic dyes sunset yellow and tartrazine in food samples. Food Chem Toxicol. 2021:156:112524. 10.1016/j.fct.2021.112524 [DOI] [PubMed] [Google Scholar]
- 42. Dey S, Nagababu BH. Applications of food color and bio-preservatives in the food and its effect on the human health. Food Chem Adv. 2022:1:100019. 10.1016/j.focha.2022.100019 [DOI] [Google Scholar]
- 43. Labiod K et al. Removal of azo dye carmoisine by adsorption process on diatomite. Adsorp Sci Technol. 2022:2022:1–17. 10.1155/2022/9517605 [DOI] [Google Scholar]
- 44. Ghalkhani M et al. Recent advances in Ponceau dyes monitoring as food colorant substances by electrochemical sensors and developed procedures for their removal from real samples. Food Chem Toxicol. 2022:161:112830. 10.1016/j.fct.2022.112830 [DOI] [PubMed] [Google Scholar]
- 45. Zarrin R, Sabzi RE. Ultrasensitive voltammetric measurement of Ponceau 4R using gold nanoparticles loaded on reduced graphene oxide on screen-printed electrode. J Iran Chem Soc. 2022:19:2973–2982. 10.1007/s13738-022-02509-8 [DOI] [Google Scholar]
- 46. Sabeen M et al. Allium cepa assay based comparative study of selected vegetables and the chromosomal aberrations due to heavy metal accumulation. Saudi J Biol Sci. 2020:27:1368–1374. 10.1016/j.sjbs.2019.12.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47. Ila HB, Husunet MT. Kromozom anormallikleri testi. In: Unal F, Yuzbasioglu D, editors. Genetik Toksikoloji. Ankara, Türkiye: Nobel Yayıncılık; 2022. pp. 153–184. [Google Scholar]
- 48. Menz J et al. Genotoxicity assessment: opportunities, challenges, and perspectives for quantitative evaluations of dose-response data. Arch Toxicol. 2023:97:2303–2328. 10.1007/s00204-023-03553-w [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49. EFSA Panel on Food Additives and Nutrient Sources Added to Food . Scientific opinion on the re-evaluation of azorubine/Carmoisine (E 122) as a food additive. EFSA J. 2009:7:1332. 10.2903/j.efsa.2009.1332 [DOI] [Google Scholar]
- 50. EFSA Panel on Food Additives and Nutrient Sources added to food . Statement on Allura red AC and other sulphonated mono azo dyes authorised as food and feed additives. EFSA J. 2013:11:3234. 10.2903/j.efsa.2013.3234 [DOI] [Google Scholar]
- 51. Sankaranarayanan N, Murthy MSS. Testing of some permitted food colors for the induction of gene conversion in diploid yeast. Mutat Res. 1979:67:309–314. 10.1016/0165-1218(79)90026-0 [DOI] [PubMed] [Google Scholar]
- 52. Ashby J, Tennant RW. Chemical structure, salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NCI/NTP. Mutat Res. 1988:204:17–115. 10.1016/0165-1218(88)90114-0 [DOI] [PubMed] [Google Scholar]
- 53. Benigni R. Analysis of the national toxicology program data on in vitro genetic toxicity tests using multivariate statistical methods. Mutagenesis. 1989:4:412–419. 10.1093/mutage/4.6.412 [DOI] [PubMed] [Google Scholar]
- 54. Zaharia D, Pavel A. The influence of certain food additives on mitosis. 4-th European cytogenesis conference. Iaşi: Dept Bioactive Substances and Medical Biotechnology, Faculty of Medical Bioengineering of Medicine and Pharmacy; 2003. [Google Scholar]
- 55. Raya SA, Aboul-Enein AM, El-Nikeety MM, Mohamed RS, Abdelwahid WMA. In vivo comet assay of food additives’ combinations and their effects on biochemical parameters in albino rats. Biointerface Res Appl Chem. 2020:24:9170–9183. [Google Scholar]
- 56. Reza MSA et al. Study of a common azo food dye in mice model: toxicity reports and its relation to carcinogenicity. Food Sci Nutr. 2019:7:667–677. 10.1002/fsn3.906 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 57. Sasaki YF et al. The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat Res Genet Toxicol Environ Mutagen. 2002:519:103–119. 10.1016/S1383-5718(02)00128-6 [DOI] [PubMed] [Google Scholar]
- 58. Jung R, Steinle D, Anliker R. A compilation of genotoxicity and carcinogenicity data on aromatic aminosulphonic acids. Food Chem Toxicol. 1992:30:635–660. 10.1016/0278-6915(92)90199-U [DOI] [PubMed] [Google Scholar]
- 59. Mishra D. Food colors and associated oxidative stress in chemical carcinogenesis. In: Chakraborti S, Ray BK, Roychowdhury S, editors. Handbook of oxidative stress in cancer: mechanistic aspects. Singapore: Springer; 2021. pp. 1–14. 10.1007/978-981-15-4501-6_182-1 [DOI] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The analyzed data sets generated during the present study are available from the corresponding author upon reasonable request.