<figure><img src="image_1.png" /></figure>

HAL
open science

# Evaluation of the toxic effects of food additives, alone or in mixture, in four human cell models

Cynthia Recoules, Mathilde Touvier, Fabrice Pierre, Marc Audebert

► To cite this version:

Cynthia Recoules, Mathilde Touvier, Fabrice Pierre, Marc Audebert. Evaluation of the toxic effects of food additives, alone or in mixture, in four human cell models. Food and Chemical Toxicology, 2025, 196, pp.115198. 10.1016/j.fct.2024.115198. hal-05178350

HAL Id: hal-05178350

https://hal.inrae.fr/hal-05178350v1

Submitted on 23 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

# Evaluation of the toxic effects of food additives, alone or in mixture, in four human cell models

Cynthia Recoules<sup>a</sup>, Mathilde Touvier<sup>b,c</sup>, Fabrice Pierre<sup>a,c</sup>, Marc Audebert<sup>a,c,*</sup>

## ARTICLE INFO

Handling Editor: Dr. Bryan Delaney

**Keywords:**
* Food additives
* Diet
* Genotoxicity
* Mixtures
* γH2AX
* pH3

## ABSTRACT

Food additives are present in more than 50% of food products. Several studies have suggested a link between the consumption of certain food additives and an increased risk of developing cancer. This study aimed to evaluate the genotoxicity of 32 additives and six mixtures identified by the NutriNet-Santé cohort as the most widely consumed. Genotoxicity screening was conducted using the γH2AX (for clastogenic compounds) and pH3 (for aneugenic compounds) biomarkers in four human cell models (colon, liver, kidney, and neurons) representing the target organs of food contaminants. The 32 compounds were categorized into five groups based on their toxicological profiles. Eight additives were cytotoxic, four promoted cell proliferation, two were genotoxic with a clastogenic mode of action, and the remaining 19 were neither cytotoxic nor genotoxic at the concentration tested. Among the six mixtures tested, three were neither cytotoxic nor genotoxic, one was cytotoxic, and two were genotoxic at the highest tested concentrations. The observed genotoxicity of the mixtures could not be attributed to the relative concentrations of the individual additives. These findings suggest the possibility of toxic synergies in mixtures and highlight the challenges of studying the combined effects of multiple substances.

## 1. Introduction

Over the past century, the consumption of 'ultra-processed' foods and their additives—substances added to food for technical purposes—has increased significantly in many countries (Touvier et al., 2023). Food additives are evaluated individually by health agencies (e.g., EFSA in Europe, FDA in the USA) based on the results of specific cytotoxicity and genotoxicity tests available at the time of their assessment. However, the toxicity of mixtures of food additives is not systematically evaluated.

During the same period, the prevalence of various metabolic diseases and cancer risks has risen (GBD, 2024). More than 75 prospective cohort studies have reported associations between the consumption of ultra-processed foods and an increased risk of cardiometabolic diseases, cancer and obesity (Adjibade et al., 2019; Beslay et al., 2020; Fiolet et al., 2018; Lane et al., 2024; Schnabel et al., 2018; Srour et al., 2019, 2020). Recent epidemiological and experimental studies also suggest that certain food additives—such as emulsifiers, nitrites, and artificial

sweeteners—may have harmful effects on chronic health conditions (Bettini et al., 2017; Chassaing et al., 2015; Crowe et al., 2019; Debras et al., 2022, 2023, 2022; Etemadi et al., 2017; He et al., 2011; Riboli et al., 2023; Salame et al., 2024; Sellem et al., 2023; Srour et al., 2022, 2023; Suez et al., 2014; Touvier et al., 2023; Viennois et al., 2017). Since 2009, the NutriNet-Santé cohort has collected unique data on the food consumption habits of over 100,000 French adults, including details on the commercial brands of industrial products consumed. This dataset enabled researchers to estimate participants' usual exposure to a wide range of food additives and their combinations (Chazelas et al., 2021). Notably, 32 food additives were identified as widely consumed, and six clusters of the population were categorized based on the primary additive mixtures they ingested: Additives found in cookies and sweet cakes; Additives commonly present in broths, meal substitutes, butter, and bread; Additives from dairy desserts, breakfast cereals, and pastries; Additives from industrial sauces and processed meats; Additives in sugary and artificially sweetened beverages; A cluster of consumers of various staple foods with low additive content.

<footnote><sup>a</sup> Toxalim, INRAE, INP-ENVT, INP-EI-Purpan, Université de Toulouse 3 Paul Sabatier, Toulouse, France</footnote>

<footnote><sup>b</sup> Université Sorbonne Paris Nord and Université Paris Cité, INSERM, INRAE, CNAM, Center of Research in Epidemiology and Statistics (CRESS), Nutritional Epidemiology Research Team (EREN), F-93017, Bobigny, France</footnote>

<footnote><sup>c</sup> Nutrition And Cancer Research Network (NACRe Network), France</footnote>

<footnote>* Corresponding author. Toxalim, INRAE, INP-ENVT, INP-EI-Purpan, Université de Toulouse 3 Paul Sabatier, Toulouse, France.
*E-mail address:* marc.audebert@inrae.fr (M. Audebert).
*URL:* http://www.inrae.fr/nacre (M. Audebert).</footnote>
<caption>**Table 1**</caption>

Intake of food additives (in mg/kg of body weight/day) according to clusters of food additive consumers in the NutriNet-Santé cohort (Chazelas et al., 2021).

<table><thead><tr><th rowspan="2">Food additive</th><th rowspan="2">% consumers</th><th>Cluster 1:</th><th>Cluster 2:</th><th>Cluster 3:</th><th>Cluster 4:</th><th>Cluster 5:</th><th>Cluster 6:</th></tr><tr><th>Consumers of additives found in cookies and sweet cakes</th><th>Consumers of additives found in broths, meal substitutes, butter, and bread</th><th>Consumers of additives found in dairy desserts, breakfast cereals and pastries</th><th>Consumers of additives found in industrial, sauces and processed meat</th><th>Consumers of additives found in sugary and artificially sweetened sodas</th><th>Consumers of various staple foods with low additive content</th></tr><tr><th></th><th></th><th>cluster_1</th><th>cluster_2</th><th>cluster_3</th><th>cluster_4</th><th>cluster_5</th><th>cluster_6</th></tr></thead><tbody><tr><td>E14xx Modified Starches</td><td>91.48</td><td>22.552</td><td>57.576</td><td>28.552</td><td>27.694</td><td>23.240</td><td>13.727</td></tr><tr><td>E330 Citric acid</td><td>91.15</td><td>41.614</td><td>36.922</td><td>30.580</td><td>46.863</td><td>33.142</td><td>21.633</td></tr><tr><td>E322 Lecithins</td><td>86.57</td><td>1.833</td><td>0.716</td><td>1.296</td><td>0.963</td><td>1.107</td><td>0.632</td></tr><tr><td>E300 Ascorbic acid</td><td>80.56</td><td>0.216</td><td>0.166</td><td>0.176</td><td>0.226</td><td>0.167</td><td>0.138</td></tr><tr><td>E415 Xanthan gum</td><td>79.74</td><td>6.865</td><td>5.868</td><td>8.107</td><td>16.910</td><td>6.591</td><td>3.855</td></tr><tr><td>E440 Pectins</td><td>78.98</td><td>3.099</td><td>4.615</td><td>3.645</td><td>4.082</td><td>5.321</td><td>2.263</td></tr><tr><td>E471 Mono- and diglycerides of fatty acids</td><td>78.07</td><td>3.555</td><td>1.949</td><td>3.706</td><td>3.803</td><td>2.963</td><td>1.844</td></tr><tr><td>E407 Carageenan</td><td>77.51</td><td>0.677</td><td>0.544</td><td>3.230</td><td>0.659</td><td>0.740</td><td>0.413</td></tr><tr><td>E250 Sodium nitrite</td><td>73.93</td><td>0.004</td><td>0.003</td><td>0.004</td><td>0.006</td><td>0.004</td><td>0.003</td></tr><tr><td>E412 Guar gum</td><td>71.00</td><td>4.065</td><td>3.324</td><td>5.002</td><td>13.492</td><td>5.035</td><td>2.744</td></tr><tr><td>E500 Sodium carbonates</td><td>67.10</td><td>77.941</td><td>14.834</td><td>20.507</td><td>16.543</td><td>20.158</td><td>12.800</td></tr><tr><td>E202 Potassium sorbate</td><td>65.81</td><td>0.315</td><td>0.231</td><td>0.429</td><td>0.719</td><td>0.368</td><td>0.178</td></tr><tr><td>E450 Diphosphates</td><td>64.36</td><td>8.957</td><td>2.176</td><td>3.235</td><td>3.009</td><td>2.794</td><td>1.830</td></tr><tr><td>E316 Sodium erythrobate</td><td>50.03</td><td>0.117</td><td>0.096</td><td>0.108</td><td>0.191</td><td>0.116</td><td>0.078</td></tr><tr><td>E301 Sodium ascorbate</td><td>49.36</td><td>0.132</td><td>0.095</td><td>0.117</td><td>0.144</td><td>0.131</td><td>0.084</td></tr><tr><td>E160c Paprika extract, capsanthin, capsorubin</td><td>49.10</td><td>0.003</td><td>0.003</td><td>0.004</td><td>0.005</td><td>0.005</td><td>0.002</td></tr><tr><td>E331 Sodium citrates</td><td>45.17</td><td>1.436</td><td>1.058</td><td>1.588</td><td>1.469</td><td>14.238</td><td>0.728</td></tr><tr><td>E224 Potassium metabisulphite</td><td>44.80</td><td>0.027</td><td>0.013</td><td>0.020</td><td>0.060</td><td>0.026</td><td>0.012</td></tr><tr><td>E503 Ammonium carbonates</td><td>43.60</td><td>35.138</td><td>3.915</td><td>6.448</td><td>5.142</td><td>7.047</td><td>3.845</td></tr><tr><td>E160a Carotenes</td><td>43.53</td><td>0.037</td><td>0.027</td><td>0.052</td><td>0.061</td><td>0.062</td><td>0.022</td></tr><tr><td>E410 Locust bean gum</td><td>38.37</td><td>1.778</td><td>0.986</td><td>2.789</td><td>4.059</td><td>2.071</td><td>1.240</td></tr><tr><td>E950 Acesulfame K</td><td>33.99</td><td>0.058</td><td>0.048</td><td>0.066</td><td>0.068</td><td>0.950</td><td>0.035</td></tr><tr><td>E270 Lactic acid</td><td>33.88</td><td>0.031</td><td>0.030</td><td>0.046</td><td>0.045</td><td>0.028</td><td>0.023</td></tr><tr><td>E120 Cochineal, Carminic acid, Carmines</td><td>33.87</td><td>0.007</td><td>0.006</td><td>0.007</td><td>0.012</td><td>0.011</td><td>0.004</td></tr><tr><td>E100 Curcumin</td><td>33.20</td><td>0.010</td><td>0.024</td><td>0.038</td><td>0.015</td><td>0.042</td><td>0.008</td></tr><tr><td>E252 Potassium nitrate</td><td>31.64</td><td>0.004</td><td>0.002</td><td>0.003</td><td>0.003</td><td>0.003</td><td>0.003</td></tr><tr><td>E150d Sulphite ammonia caramel</td><td>28.79</td><td>1.621</td><td>0.556</td><td>1.382</td><td>1.446</td><td>23.721</td><td>0.855</td></tr><tr><td>E420 Sorbitols</td><td>27.96</td><td>1.802</td><td>0.594</td><td>0.918</td><td>0.930</td><td>0.949</td><td>0.574</td></tr><tr><td>E951 Aspartame</td><td>27.73</td><td>0.103</td><td>0.112</td><td>0.113</td><td>0.120</td><td>1.608</td><td>0.089</td></tr><tr><td>E422 Glycerol</td><td>25.49</td><td>7.658</td><td>1.837</td><td>2.562</td><td>2.549</td><td>2.550</td><td>1.501</td></tr><tr><td>E161b Lutein</td><td>25.34</td><td>0.010</td><td>0.010</td><td>0.007</td><td>0.013</td><td>0.020</td><td>0.005</td></tr><tr><td>E621 Monosodium glutamate</td><td>&lt;20</td><td>45.461</td><td>60.122</td><td>30.343</td><td>45.719</td><td>20.545</td><td>31.523</td></tr><tr><td>Sum</td><td></td><td>267.126</td><td>198.458</td><td>155.080</td><td>197.020</td><td>175.753</td><td>102.691</td></tr></tbody></table>

In this study, we investigated these additives both individually and in combinations that mimicked the mixtures observed in 'real-life' scenarios within the NutriNet-Santé cohort (Chazelas et al., 2021). Table 1 lists the 32 food additives consumed daily, ranked from the most to the least consumed, along with their proportions in the six mixtures.

To better understand the potential toxic effects of these food additives and their mixtures on human health, we screened the effects of these substances using four different human cell lines. We selected two cell lines with metabolic capacity: LS-174T (colon) and HepG2 (liver), as

well as two cell lines without metabolic capacity: ACHN (kidney) and SH-SY5Y (neuron) (Khoury et al., 2016).

Our study focused on two novel sensitive genotoxicity biomarkers, γH2AX and pH3 (Kopp et al., 2019). γH2AX, a marker related to histone H2AX phosphorylation, is an early and sensitive genotoxicity biomarker induced by various types of DNA damage caused by clastogenic compounds. Meanwhile, pH3, a marker of histone H3 phosphorylation, is a sensitive genotoxicity biomarker associated with cells blocked in mitosis notably due to aneugenic compounds.
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** *In vitro* cytotoxicity and genotoxicity of the E450 additivetested for γH2AX and pH3 after 24H treatment in the HepG2 cell line. Cytotoxicity is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (\*\*P ≤ 0.01).</figcaption></figure>

We exposed the human cells to five concentrations of the 32 additives, individually or in mixtures, for one day to assess cytotoxicity and genotoxicity using the γH2AX/pH3 assay. This strategy has been successfully applied previously to other food contaminants (Graillot et al., 2012; Kopp et al., 2020). For single-additive treatments, we observed some instances of cytotoxicity and/or genotoxicity, with evidence suggesting the possibility of toxic synergies in mixtures.

## 2. Materials and methods

### 2.1. Chemicals and reagents

The additives were sourced from several suppliers in France: Cuisine Addict [carmine acid (E120 – European code; CAS RN® 1260-17-9), lutein (E161b; CAS RN® 127-40-2)], Louis Francois [modified starches (E1442; CAS RN® 68130-14-3), citric acid (E330; CAS RN® 77-92-9), lecithins (E322; CAS RN® 8002-43-5), ascorbic acid (E300; CAS RN® 50-81-7), xanthan gum (E415; CAS RN® 11138-66-2), pectins (E440; CAS RN® 9000-69-5), mono- and diglycerides of fatty acids (E471; CAS RN® 123-94-4), carrageenan (E407; CAS RN® 9000-07-1), sodium carbonates (E500; CAS RN® 144-55-8), sodium citrate (E331; CAS RN® 6132-04-3), ammonium carbonates (E503; CAS RN® 506-87-6), sorbitols (E420; CAS RN® 50-70-4), glycerol (E422; CAS RN® 58-81-5)], Toque de chef [guar gum (E412; CAS RN® 9000-30-0), potassium sorbate (E202; CAS RN® 4634-61-5), diphosphates (E450; CAS RN® 7758-16-9), locust bean gum (E410; CAS RN® 9000-40-2), potassium nitrate (E252; CAS RN® 7757-79-1)], Jardins des épices [sodium nitrite (E250; CAS RN® 7757-79-1), paprika extract, capsanthin, capsorubin (E160c; CAS RN® 470-38-2), curcumin (E100; CAS RN® 458-37-7), sulfite ammonia caramel (E150d; CAS RN® 8028-89-5), monosodium glutamate (E621; CAS RN® 142-47-2)], Dumortier [sodium ascorbate (E301; CAS RN® 134-03-2), sodium erythorbate (E316; CAS RN® 7378-23-6)], and Sigma-Aldrich [potassium disulfite (E224; CAS RN® 16731-55-8), carotenes (E160a; CAS RN® 7235-40-7), acesulfame K (E950; CAS RN® 55589-62-3), lactic acid (E270; CAS RN® 50-21-5), aspartame (E951; CAS RN® 22839-47-0)]. All additives were prepared in phosphate-buffered saline (PBS).

The positive genotoxic chemicals benzo[a]pyrene (BaP; CAS 50-32-8), etoposide (ETO; CAS RN® 33419-42-0), and nocodazole (NOCO; CAS RN® 31430-18-9) were purchased from Sigma-Aldrich (France) and dissolved in dimethyl sulfoxide (DMSO). Additional reagents, including penicillin, trypsin, PBS, RNase A, and Triton X-100, were also obtained from Sigma-Aldrich (France). The blocking solution (Max-block Blocking Medium) was purchased from Active Motif (Belgium).

### 2.2. Cell culture

In this study, we used four cell lines. The LS-174T (colon) and HepG2 (liver) cell lines possess metabolic properties, whereas the ACHN (kidney) and SH-SY5Y (neuronal) cell lines lack bioactivation capacity. Briefly, LS-174T, HepG2, and ACHN cells were cultured in growth medium (MEM-alpha supplemented with 10% serum and 1% penicillin/streptomycin), while SH-SY5Y cells were cultured in growth medium (DMEM-F12 supplemented with 10% serum and 1% penicillin/streptomycin). After reaching confluence, the cells were trypsinized, counted, and seeded at a density of 32,000 cells per well in Greiner 96-well transparent black-bottom plates, which are compatible with fluorescence analysis.

### 2.3. Cell treatments

Cells were treated with the 32 additives individually for 24 h at five different concentrations (0.1, 0.3, 1, 3, and 10 mg/mL), as recommended for genotoxicity studies. To create the six food additive mixtures, the 32 individual additive solutions were combined in specific proportions to replicate the intake patterns observed in the six clusters/mixtures identified in the NutriNet-Santé study (Table 1).

The intake data for each additive in each mixture, quantified in NutriNet-Santé (in mg/kg of body weight/day), were used to estimate the daily exposure per person. The calculation involved multiplying the intake by 60 (assuming an average body weight of 60 kg per person) and dividing by 5 (the average blood volume in liters per person) and 1000 (to convert liters to milliliters). A 100X concentrated stock solution was prepared for each mixture.

Cells were treated with a 10-fold dilution of the stock solution, followed by serial dilutions. All dilutions were prepared in PBS. The 1X *in vitro* concentration tested approximates the *in vivo* dose resulting from the current mean consumption of these additives by participants in the nutrition-health cohort.

### 2.4. Genotoxicity analysis with the γH2AX/pH3 assay

The γH2AX/pH3 assay was performed as described in previous publications (Khoury et al., 2016; Kopp et al., 2019; Recoules et al., 2024). After treatment, cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by a PBS wash. Permeabilization was performed using 0.2% Triton X-100 in PBS for 5 min, followed by a PST solution wash (0.2% Triton X-100 and 0.2% serum in PBS). Cells were then blocked for 1 h at room temperature with MAXblock Blocking Medium supplemented with 0.1% RNase and subsequently washed with PST.

The primary antibody solution, containing rabbit monoclonal anti-γH2AX antibody (Clone 20E3, Cell Signaling) and mouse monoclonal anti-pH3 antibody (Clone CM312, Sigma), was incubated with the cells for 2 h. This was followed by three consecutive 5-min washes with PST solution. Secondary detection was performed using fluorescently labeled antibodies: CF770 goat antibody and CF680 donkey antibody (Biotium). Sybergold (Invitrogen) was added during the secondary antibody incubation for 1 h to label DNA. Finally, three consecutive washes were performed. DNA, γH2AX, and pH3 fluorescence signals were quantified simultaneously using the Sapphire Biomolecular Imager (Azure Biosystems, USA).

The γH2AX/pH3 assay enabled simultaneous evaluation of cytotoxicity and genotoxicity within a single experiment. Cytotoxicity was assessed by comparing the DNA content of treated cells—correlated to cell count—to the DNA content of PBS-treated control cells, expressed as a percentage or relative cell count (% RCC). Genotoxicity was determined by calculating the change in phosphorylation levels of the histones. This was achieved by dividing the γH2AX and pH3 fluorescence intensities in treated wells by the intensities in untreated wells.

We also assessed the reactivity and autofluorescence of food color
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** *In vitro* cytotoxicity and genotoxicity of six additives from antioxidant tested for γH2AX and pH3 after one treatment in the HepG2 cell line. Panel A: E330; Panel B: E300; Panel C: E322; Panel D: E316; Panel E: E331; Panel F: E301. Cytotoxicity is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (*P* ≤ 0.05; **P** ≤ 0.01).</figcaption></figure>

additives (E100, E161b, E120, E160c, E160a, and E150d) at 680 nm and 770 nm. For this, we evaluated the additives in wells without primary antibodies to determine background autofluorescence. The background fluorescence values were then subtracted from the results obtained in wells containing primary antibodies.

## 2.5. Statistical analysis

The observations from at least three independent experiments are presented as the mean ± standard error of the mean (SEM). Statistical significance of biomarker increases after treatment, compared to the vehicle (PBS) control, was determined using a two-sided Student's t-test (*P* ≤ 0.05; **P** ≤ 0.01). A result was considered positive if the measured biomarker increased by more than 1.5-fold and cytotoxicity was less

than 50% relative to the PBS control. If cytotoxicity exceeded 50%, biomarkers data were not displayed. Changes in cytotoxicity levels were also statistically analyzed by comparing treated samples to the PBS control using a two-sided Student's t-test (*P* ≤ 0.05; **P** ≤ 0.01).

## 3. Results

The assays were conducted with a maximum concentration of 10 mg/mL for each additive tested alone. Each experiment included positive and negative controls. BaP (1 µM), a bioactivated genotoxin, induced a significant increase in γH2AX levels after 24 h of treatment in the LS-174T and HepG2 cell lines, as expected in metabolically competent cells. ETO (10 µM) and NOCO (1 µM) treatments significantly increased γH2AX and pH3 levels, respectively, in all tested cell lines
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** *In vitro* cytotoxicity and genotoxicity of five preservative additives tested for γH2AX and pH3 after one treatment in the HepG2 cell line. Panel A: E252; Panel B: E202; Panel C: E250; Panel D: E224; Panel E: E270. Cytotoxicity is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (*P* ≤ 0.05; **P** ≤ 0.01).</figcaption></figure>

(Supplementary Data 1).

### 3.1. Texture agents, sweeteners, and flavor enhancers

Among texture agents, E1442, E415, E440, E471, E407, E412, E410, and E422 showed no significant changes in γH2AX or pH3 levels in HepG2 cells (Supplementary Data 2). E450, however, caused a

significant decrease in cell viability at 10 mg/mL in HepG2 cells (Fig. 1), a result replicated in LS-174T, ACHN, and SH-SY5Y cells (Supplementary Data 3, 8, and 13). Sweeteners E950, E420, and E951, as well as flavor enhancer E621, had no significant effects on γH2AX or pH3 biomarkers in HepG2 cells (Supplementary Data 2) or the other cell lines (Supplementary Data 3, 8, and 13).
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** *In vitro* cytotoxicity and genotoxicity of five acidity regulator additives tested for γH2AX and pH3 after one treatment in the HepG2 cell line. Panel A: E500; Panel B: E503. Cytotoxicity is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (\*\*P ≤ 0.01).</figcaption></figure>

### 3.2. Antioxidants

Antioxidants yielded three types of results. E330 and E300 significantly increased cell viability at 10 mg/mL in HepG2 cells (Fig. 2A and B). E322, E316, and E331 had no significant effects (Fig. 2C, D, 2E). E301 caused a significant decrease in cell viability at 10 mg/mL in HepG2 cells (Fig. 2F), with similar effects observed in LS-174T, ACHN, and SH-SY5Y cells (Supplementary Data 4, 9, and 14).

### 3.3. Preservatives

Preservatives E252 and E202 did not significantly alter γH2AX or pH3 levels in HepG2 cells (Fig. 3A and B). E270 increased cell viability at 10 mg/mL (Fig. 3E), while E250 decreased cell viability at the same concentration (Fig. 3C), with comparable effects in LS-174T, ACHN, and SH-SY5Y cells (Supplementary Data 5, 10, and 15).

### 3.4. Acidity regulators

E500 showed no significant toxic effects in HepG2 cells (Fig. 4A) or the other cell models (Table 2). In contrast, E503 significantly increased γH2AX levels at 3 mg/mL and decreased cell viability at 10 mg/mL in HepG2 cells (Fig. 4B). Similar increases in γH2AX were observed in LS-174T and SH-SY5Y cells at 3 and 10 mg/mL, respectively (Supplementary Data 6 and 16), alongside decreased cell viability in ACHN cells (Supplementary Data 11).

### 3.5. Dyes

Dyes E100, E161b, E160c, E160a, and E150d did not significantly affect γH2AX or pH3 biomarkers in HepG2 cells (Fig. 5A, B, 5D, 5E, 5F). However, E160c decreased cell viability at 3 mg/mL (Fig. 5D). E120 significantly increased γH2AX levels from 1 mg/mL to 10 mg/mL and reduced cell viability at 3 mg/mL in HepG2 cells (Fig. 5C), with similar results in LS-174T and SH-SY5Y cells (Supplementary Data 7 and 17).

### 3.6. Effect of food additive mixtures

In HepG2 cells, mixture 1 (M1) increased γH2AX and pH3 biomarkers and decreased cell viability at the highest 10X concentration

tested (Fig. 6A). A decrease in cell viability was also observed at this concentration in LS-174T, ACHN, and SH-SY5Y cells (Supplementary Data 18, 21, and 24).

Mixture 2 (M2) increased γH2AX levels at the 10X concentration in HepG2 and LS-174T cells (Fig. 6B and Supplementary Data 18) and reduced cell viability in LS-174T, ACHN, and SH-SY5Y cells (Supplementary Data 18, 21, and 24).

No cytotoxicity or genotoxicity was observed for mixtures M3, M4, or M6 in any cell line at any concentration tested after 24 h of treatment (Fig. 7A, B, 7C and Supplementary Data 19, 22, and 25).

Mixture 5 (M5) decreased cell viability from the 3X concentration in HepG2 and ACHN cells (Fig. 8 and Supplementary Data 23), with no effects observed in LS-174T and SH-SY5Y cells (Supplementary Data 20 and 26).

## 4. Discussion

The aim of this study was to compare the toxicity potential of 32 widely consumed food additives, both individually and in mixtures (representative of human exposure), in four human cell lines (LS-174T, HepG2, ACHN, and SH-SY5Y) following a 24-h treatment.

Each additive was tested alone at five concentrations over a 24-h period. Of the 32 additives, 19 showed no cytotoxicity or genotoxicity (E1442, E322, E415, E440, E471, E407, E412, E500, E410, E950, E420, E951, E422, E161b, E621, E160c, E150d, and E160a). Seven additives demonstrated cytotoxicity (E450, E100, E250, E202, E316, E301, and E331), four enhanced cell proliferation (E330, E300, E224, and E270), and two were genotoxic with a clastogenic mode of action (E120 and E503) (Table 2).

For most additives within the following groups—texture agents (E1442, E415, E440, E471, E407, E412, E450, E410, and E422), antioxidants (E330, E322, E300, E316, E301, and E331), preservatives (E250, E202, E224, E270, and E252), acidity regulators (E500), dyes (E160c, E160a, E100, E150d, and E161b), sweeteners (E950, E420, and E951), and flavor enhancers (E621)—genotoxicity results were negative. These findings align with previous literature (Authority, 2014; Çadirci et al., 2020; Dosay-Akbulut, 2020; Kirkland et al., 2005; Kirkland and Gatehouse, 2015; Yamada and Honma, 2018), except for E503 and E120. These two additives were genotoxic in HepG2, LS-174T, and SH-SY5Y cells but not in ACHN cells. Previous studies have reported
<caption>**Table 2**</caption>

**Summary results of genotoxicity and cytotoxicity for the 32 additives in the four cell lines.** The γH2AX/pH3 assay results in HepG2, LS-174T, ACHN and SH-SY5Y cell lines. Pink and upward arrows represent an increase in histone phosphorylation compared to the control. Green and downward arrows represent a decrease in histone phosphorylation compared to the control. Blue and upward arrows represent an increase in cell viability. Concentrations (in µM) correspond to the lowest effective concentration (LOEC) observed. Clear boxes indicate no biomarker variation.

<table><thead><tr><th rowspan="2">Name</th><th colspan="3">HepG2</th><th colspan="3">LS-174T</th><th colspan="3">ACHN</th><th colspan="3">SH-SY5Y</th></tr><tr><th>γH2AX</th><th>pH3</th><th>% RCC</th><th>γH2AX</th><th>pH3</th><th>% RCC</th><th>γH2AX</th><th>pH3</th><th>% RCC</th><th>γH2AX</th><th>pH3</th><th>% RCC</th></tr></thead><tbody><tr><td colspan="13"><strong>Texture agent</strong></td></tr><tr><td>E1442 Modified Starches</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E415 Xanthan Gum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E440 Pectins</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E471 Mono-and Diglycerides of fatty acids</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E407 Carrageenan</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E412 Guar Gum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E450 Diphsphates</td><td>-</td><td>γ 10</td><td>γ 10</td><td>-</td><td>γ 10</td><td>-</td><td>γ 1</td><td>γ 10</td><td>-</td><td>-</td><td>γ 10</td><td>-</td></tr><tr><td>E410 Locust bean gum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E422 Glycerol</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td colspan="13"><strong>Antioxidant</strong></td></tr><tr><td>E330 Citric acid</td><td>γ 3</td><td>γ 3</td><td>γ 10</td><td>γ 3</td><td>γ 3</td><td>γ 10</td><td>γ 3</td><td>γ 3</td><td>γ 10</td><td>γ 3</td><td>γ 3</td><td>γ 10</td></tr><tr><td>E322 Lecithins</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E300 Ascorbic acid</td><td>γ 10</td><td>-</td><td>γ 10</td><td>γ 10</td><td>-</td><td>γ 10</td><td>γ 10</td><td>-</td><td>γ 10</td><td>γ 10</td><td>-</td><td>γ 10</td></tr><tr><td>E316 Sodium erythorbate</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>γ 10</td><td>-</td><td>-</td><td>-</td><td>-</td><td>γ 10</td></tr><tr><td>E301 Sodium ascorbate</td><td>-</td><td>-</td><td>γ 10</td><td>-</td><td>γ 10</td><td>-</td><td>γ 3</td><td>-</td><td>γ 3</td><td>-</td><td>γ 3</td><td>γ 3</td></tr><tr><td>E331 Sodium citrates</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>γ 10</td><td>-</td><td>γ 10</td><td>-</td><td>γ 10</td><td>γ 10</td><td>γ 10</td></tr><tr><td colspan="13"><strong>Preservative</strong></td></tr><tr><td>E250 Sodium nitrite</td><td>-</td><td>-</td><td>γ 10</td><td>-</td><td>-</td><td>γ 10</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E202 Potassium sorbate</td><td>-</td><td>-</td><td>-</td><td>γ 10</td><td>-</td><td>γ 10</td><td>-</td><td>γ 10</td><td>-</td><td>γ 10</td><td>γ 10</td><td>γ 10</td></tr><tr><td>E224 Potassium metabisulphite</td><td>γ 3</td><td>γ 3</td><td>-</td><td>γ 3</td><td>γ 3</td><td>γ 3 et γ 10</td><td>γ 10</td><td>γ 3</td><td>γ 3</td><td>γ 1</td><td>γ 1</td><td>γ 3 et γ 10</td></tr><tr><td>E270 Lactic acid</td><td>γ 3</td><td>γ 10</td><td>γ 10</td><td>γ 3</td><td>γ 3</td><td>γ 10</td><td>γ 3</td><td>γ 3</td><td>γ 3 et γ 10</td><td>γ 3</td><td>γ 3</td><td>γ 3</td></tr><tr><td>E252 Potassium nitrate</td><td>-</td><td>-</td><td>-</td><td>γ 10</td><td>γ 10</td><td>-</td><td>γ 10</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td colspan="13"><strong>Acidity regulator</strong></td></tr><tr><td>E500 Sodium carbonates</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E503 Ammonium carbonates</td><td>γ 3</td><td>-</td><td>γ 10</td><td>γ 3</td><td>γ 3</td><td>γ 10</td><td>γ 10</td><td>γ 10</td><td>γ 10</td><td>γ 10</td><td>γ 10</td><td>γ 10</td></tr><tr><td colspan="13"><strong>Dye</strong></td></tr><tr><td>E160c Paprika extract, capsanthin, capsorubin</td><td>-</td><td>-</td><td>γ 3</td><td>-</td><td>-</td><td>γ 3</td><td>-</td><td>-</td><td>γ 3</td><td>-</td><td>-</td><td>γ 3</td></tr><tr><td>E160a Carotenes</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>γ 1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E120 Cochineal, Carminic acid, Carmines</td><td>γ 1</td><td>-</td><td>γ 1</td><td>γ 1</td><td>-</td><td>γ 3</td><td>-</td><td>-</td><td>γ 3</td><td>γ 10</td><td>-</td><td>γ 3</td></tr><tr><td>E100 Curcumin</td><td>-</td><td>-</td><td>γ 1</td><td>γ 1</td><td>-</td><td>γ 1</td><td>γ 3</td><td>-</td><td>γ 3</td><td>-</td><td>-</td><td>γ 10</td></tr><tr><td>E150d Sulphite ammonia caramel</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E161b Lutein</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td colspan="13"><strong>Sweetening agent</strong></td></tr><tr><td>E950 Acesulfame K</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E420 Sorbitols</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E951 Aspartame</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td colspan="13"><strong>Flavor Enhancer</strong></td></tr><tr><td>E621 Monosodium glutamate</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** *In vitro* cytotoxicity and genotoxicity of six dye additives tested for γH2AX and pH3 after one treatment in the HepG2 cell line. Panel A: E100; Panel B: E161b; Panel C: E120; Panel D: E160c; Panel E: E160a; Panel F: E150d. Cytotoxicity is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (*P* ≤ 0.05; **P* ≤ 0.01).</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** *In vitro* cytotoxicity and genotoxicity of two mixtures tested for γH2AX and pH3 after one treatment in the HepG2 cell line. Panel A: M1; Panel B: M2. Cytotoxicity is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (*P* ≤ 0.05; **P** ≤ 0.01).</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 7.** *In vitro* cytotoxicity and genotoxicity of three mixtures tested for γH2AX and pH3 after one treatment in the HepG2 cell line. Panel A: M3; Panel B: M4; Panel C: M6. Cytotoxicity is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3).</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 8.** *In vitro* cytotoxicity and genotoxicity for the M5 mixture tested for γH2AX and pH3 after one treatment in the HepG2 cell line. Cytotoxicity is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (\*\*P ≤ 0.01).</figcaption></figure>

negative results for E503 and E120 in standard *in vitro* genotoxicity says (EFSA, 2015; Yamada and Honma, 2018). This study demonstrated the efficiency of the γH2AX/pH3 method for genotoxicity screening in the context of food additives risk assessment.

Six mixtures of the 32 food additives were tested at five concentrations over a 24-h treatment. Three mixtures (M3, M4, and M6) showed no cytotoxicity or genotoxicity, while one mixture (M5) was cytotoxic. Two mixtures (M1 and M2) were genotoxic at the highest tested concentrations (Table 3).

Mixture M1 induced significant γH2AX levels in HepG2 and SH-SY5Y cells but not in ACHN or LS-174T cells. M1 contained higher concentrations of E503, E1442, E330, E500, and E621. Genotoxicity observed at 10X concentration (containing 4 mg/mL of E503) was consistent with E503's individual positive result at 3 mg/mL, suggesting that E503 may drive the genotoxic effect of M1.

In the mixture M2, genotoxicity was observed in HepG2 and LS-174T cells but not in ACHN or SH-SY5Y cells, suggesting a requirement for metabolic activation. Differences between cell models may also reflect variations in antioxidant defenses and DNA repair capacities (Khoury et al., 2016). The M2 mixture, which contained high levels of E1442, E330, E500, and E621, exhibited a synergistic effect, as these individual additives did not induce γH2AX or pH3 biomarkers.

For the Mixture M5, Cytotoxicity was observed starting at the 3X concentration in HepG2 and ACHN cells. The higher concentrations of E330 and E331 in M5 may explain this effect, as both demonstrated cytotoxicity when tested individually.

This study employed a 24-h treatment protocol as recommended for regulatory genotoxicity testing. However, long-term or repeated exposure protocols may better assess the genotoxic potential of certain chemicals (Recoules et al., 2024). Future experiments using extended treatment periods or 3D human cell models could provide valuable insights.

The primary goal was to investigate the genotoxicity of 32 commonly consumed food additives and six mixtures corresponding to dietary exposure in France. A key strength of this study lies in its use of human exposure data to design realistic additive mixtures. While our findings indicate the potential toxicity of some combinations, further research is needed to determine whether the *in vitro* concentrations used are predictive of a possible human health risks (Beal et al., 2024).

This study employed four human cell lines representing major target organs to evaluate the potential harmful effects of food additive mixtures commonly found in the French diet. We observed that two mixtures induced genotoxicity and one mixture caused cytotoxicity in a concentration-dependent manner. These findings emphasize the limitations of risk assessments based solely on individual compound effects and highlight the importance of evaluating chemical mixtures.

### CRediT authorship contribution statement

**Cynthia Recoules:** Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Conceptualization. **Mathilde Touvier:** Writing – review & editing, Project administration, Funding acquisition. **Fabrice Pierre:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization. **Marc Audebert:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

<caption>**Table 3**</caption>

Summary results of genotoxicity and cytotoxicity of the four cell lines for the six mixtures. The γH2AX/pH3 assay results in HepG2, LS-174T, ACHN and SH-SY5Y cell lines. Pink and upward arrows represent an increase in histone phosphorylation compared to the control. Green and downward arrows represent a decrease in histone phosphorylation compared to the control. Concentrations (in µM) correspond to the lowest effective concentration (LOEC) observed. Clear boxes indicate no biomarker variation.

<table><thead><tr><th rowspan="2">Name</th><th colspan="3">HepG2</th><th colspan="3">LS-174T</th><th colspan="3">ACHN</th><th colspan="3">SH-SY5Y</th></tr><tr><th>γH2AX</th><th>pH3</th><th>% RCC</th><th>γH2AX</th><th>pH3</th><th>% RCC</th><th>γH2AX</th><th>pH3</th><th>% RCC</th><th>γH2AX</th><th>pH3</th><th>% RCC</th></tr></thead><tbody><tr><td>M1</td><td>▲ 10</td><td>▲ 10</td><td>▲ 10</td><td>-</td><td>-</td><td>▲ 10</td><td>▲ 10</td><td>-</td><td>▲ 10</td><td>▲ 10</td><td>▲ 10</td><td>▲ 10</td></tr><tr><td>M2</td><td>▲ 10</td><td>-</td><td>-</td><td>▲ 10</td><td>-</td><td>▲ 10</td><td>-</td><td>-</td><td>▲ 10</td><td>-</td><td>▲ 10</td><td>▲ 10</td></tr><tr><td>M3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M5</td><td>-</td><td>-</td><td>▲ 3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>▲ 3</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M6</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>
## Acknowledgements

This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No 864219, ADDITIVES), the French National Cancer Institute (INCa_14059), the French Ministry of Health (arrêté 29.11.19) the IdEx Université de Paris (ANR-18-IDEX-0001), and a Bettencourt-Schuelius Foundation Research Prize 2021. This project was awarded the NACRe (French network for Nutrition And Cancer Research) Partnership Label. Researchers were independent from funders. Funders had no role in the study design, the collection, analysis, and interpretation of data, the writing of the report, and the decision to submit the article for publication.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fct.2024.115198.

## Data availability

Data will be made available on request.

## References

Adjibade, M., Julia, C., Allès, B., Touvier, M., Lemogne, C., Srour, B., Hercberg, S., Galan, P., Assmann, K.E., Kesse-Guyot, E., 2019. Prospective association between ultra-processed food consumption and incident depressive symptoms in the French NutriNet-Santé cohort. BMC Med. 17, 78. https://doi.org/10.1186/s12916-019-1312-y.

Authority, E.F.S., 2014. Refined exposure assessment for curcumin (E 100). EFSA J. 12, 3876. https://doi.org/10.2903/j.efsa.2014.3876.

Beal, M., Habauzit, D., Khoury, L., Audebert, M., 2024. Human next-generation risk assessment of trichothecene toxicity. Food Chem. Toxicol. 192, 114916. https://doi.org/10.1016/j.fct.2024.114916, 2024 Oct.

Beslay, M., Srour, B., Méjean, C., Allès, B., Fiolet, T., Debras, C., Chazelas, E., Deschasaux, M., Wendeu-Foyet, M.G., Hercberg, S., Galan, P., Monteiro, C.A., Deschamps, V., Calixto Andrade, G., Kesse-Guyot, E., Julia, C., Touvier, M., 2020. Ultra-processed food intake in association with BMI change and risk of overweight and obesity: a prospective analysis of the French NutriNet-Santé cohort. PLoS Med. 17, e1003256. https://doi.org/10.1371/journal.pmed.1003256.

Bettini, S., Boutet-Robinet, E., Cartier, C., Coméra, C., Gaultier, E., Dupuy, J., Naud, N., Taché, S., Grysan, P., Reguer, S., Thieriet, N., Réfrégiers, M., Thiaudière, D., Cravedi, J.-P., Carrière, M., Audinot, J.-N., Pierre, F.H., Guzylack-Piriou, L., Houdeau, E., 2017. Food-grade TiO2 impairs intestinal and systemic immune homeostasis, initiates preneoplastic lesions and promotes aberrant crypt development in the rat colon. Sci. Rep. 7, 40373. https://doi.org/10.1038/srep40373.

Çadirci, K., Özdemir Tozlu, Ö., Türkez, H., Mardinoğlu, A., 2020. The in vitro cytotoxic, genotoxic, and oxidative damage potentials of the oral artificial sweetener aspartame on cultured human blood cells. Turk. J. Med. Sci. 50, 448-454. https://doi.org/10.3906/sag-2001-113.

Chassaing, B., Koren, O., Goodrich, J.K., Poole, A.C., Srinivasan, S., Ley, R.E., Gewirtz, A. T., 2015. Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome. Nature 519, 92-96. https://doi.org/10.1038/nature14232.

Chazelas, E., Druesne-Pecollo, N., Esseddik, Y., de Edelenyi, F.S., Agaësse, C., De Sa, A., Lutchia, R., Rebouillat, P., Srour, B., Debras, C., Wendeu-Foyet, G., Huybrechts, I., Pierre, F., Coumoul, X., Julia, C., Kesse-Guyot, E., Allès, B., Galan, P., Hercberg, S., Deschasaux-Tanguy, M., Touvier, M., 2021. Exposure to food additive mixtures in 106,000 French adults from the NutriNet-Santé cohort. Sci. Rep. 11, 19680. https://doi.org/10.1038/s41598-021-98496-e.

Crowe, W., Elliott, C.T., Green, B.D., 2019. A review of the in vivo evidence investigating the role of nitrite exposure from processed meat consumption in the development of colorectal cancer. Nutrients 11, 2673. https://doi.org/10.3390/nu11112673.

Debras, C., Chazelas, E., Sellem, L., Porcher, R., Druesne-Pecollo, N., Esseddik, Y., de Edelenyi, F.S., Agaësse, C., De Sa, A., Lutchia, R., Fezeu, L.K., Julia, C., Kesse-Guyot, E., Allès, B., Galan, P., Hercberg, S., Deschasaux-Tanguy, M., Huybrechts, I., Srour, B., Touvier, M., 2022. Artificial sweeteners and risk of cardiovascular diseases: results from the prospective NutriNet-Santé cohort. BMJ 378, e071204. https://doi.org/10.1136/bmj-2022-071204.

Debras, C., Deschasaux-Tanguy, M., Chazelas, E., Sellem, L., Druesne-Pecollo, N., Esseddik, Y., Szabo de Edelenyi, F., Agaësse, C., De Sa, A., Lutchia, R., Julia, C., Kesse-Guyot, E., Allès, B., Galan, P., Hercberg, S., Huybrechts, I., Cosson, E., Tatulashvili, S., Srour, B., Touvier, M., 2023. Artificial sweeteners and risk of type 2 diabetes in the prospective NutriNet-santé cohort. Diabetes Care 46, 1681-1690. https://doi.org/10.2337/dc23-0206.

Dosay-Akbulut, M., 2020. Determination of DNA damage caused by food additives using comet assay method : food additives DNA damage via comet assay. Prog. Nutr. 22, e2002071. https://doi.org/10.23751/pn.v22i4.9095.

EFSA, 2015. Scientific Opinion on the re-evaluation of cochineal, carminic acid, carmines (E 120) as a food additive. EFSA J. 13, 4288. https://doi.org/10.2903/j.efsa.2015.4288.

Emetadi, A., Sinha, R., Ward, M.H., Graubard, B.I., Inoue-Choi, M., Dawsey, S.M., Abnet, C.C., 2017. Mortality from different causes associated with meat, heme iron, nitrates, and nitrites in the NIH-AARP Diet and Health Study: population based cohort study. BMJ j1957. https://doi.org/10.1136/bmj.j1957.

Fiolet, T., Srour, B., Sellem, L., Kesse-Guyot, E., Allès, B., Méjean, C., Deschasaux, M., Fassier, P., Latino-Martel, P., Beslay, M., Hercberg, S., Lavalette, C., Monteiro, C.A., Julia, C., Touvier, M., 2018. Consumption of ultra-processed foods and cancer risk: results from NutriNet-Santé prospective cohort. BMJ 360, k322. https://doi.org/10.1136/bmj.k322.

GBD 2021 Diseases and Injuries Collaborators, 2024. Global incidence, prevalence, years lived with disability (YLDs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries in 204 countries and territories and 811 subnational locations, 1990-2021: a systematic analysis for the Global Burden of Disease Study 2021. Lancet 403, 2133-2161. https://doi.org/10.1016/S0140-6736 (24)00757-8.

Grailott, V., Takakura, N., Hegarat, L.L., Fessard, V., Audebert, M., Cravedi, J., 2012. Genotoxicity of pesticide mixtures present in the diet of the French population. Environ. Mol. Mutagen. 53, 173-184. https://doi.org/10.1002/em.21676.

He, K., Du, S., Xun, P., Sharma, S., Wang, H., Zhai, F., Popkin, B., 2011. Consumption of monosodium glutamate in relation to incidence of overweight in Chinese adults: China Health and Nutrition Survey (CHNS). Am. J. Clin. Nutr. 93, 1328-1336. https://doi.org/10.3945/ajcn.110.008870.

Khoury, L., Zalko, D., Audebert, M., 2016. Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening. Mutagenesis 31, 83-96. https://doi.org/10.1093/mutage/gev058.

Kirkland, D., Aardema, M., Henderson, L., Müller, L., 2005. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: I. Sensitivity, specificity and relative predictivity. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 584, 1-256. https://doi.org/10.1016/j.mrgentox.2005.02.004.

Kirkland, D., Gatehouse, D., 2015. Aspartame: a review of genotoxicity data. Food Chem. Toxicol. 84, 161-168. https://doi.org/10.1016/j.fct.2015.08.021.

Kopp, B., Khoury, L., Audebert, M., 2019. Validation of the γH2AX biomarker for genotoxicity assessment: a review. Arch. Toxicol. 93, 2103-2114. https://doi.org/10.1007/s00204-019-02511-9.

Kopp, B., Le Hegarat, L., Audebert, M., 2020. Differential toxic effects of food contaminant mixtures in HepaRG cells after single or repeated treatments. Mutat. Res. Genet. Toxicol. Environ. Mutagen 850-851, 503161. https://doi.org/10.1016/j.mrgentox.2020.503161.

Lane, M.M., Gamage, E., Du, S., Ashtree, D.N., McGuinness, A.J., Gauci, S., Baker, P., Lawrence, M., Rebholz, C.M., Srour, B., Touvier, M., Jacka, F.N., O'Neil, A., Segasby, T., Marx, W., 2024. Ultra-processed food exposure and adverse health outcomes: umbrella review of epidemiological meta-analyses. BMJ 384, e077310. https://doi.org/10.1136/bmj-2023-077310.

Recoules, C., Mirey, G., Audebert, M., 2024. Effect of cell treatment procedures on in vitro genotoxicity assessment. Arch. Toxicol. https://doi.org/10.1007/s00204-024-03690-w.

Riboli, E., Beland, F.A., Lachenmeier, D.W., Marques, M.M., Phillips, D.H., Schernhammer, E., Afghan, A., Assunção, R., Caderni, G., Corton, J.C., de Aragão Umbuzeiro, G., de Jong, D., Deschasaux-Tanguy, M., Hodge, A., Ishihara, J., Levy, D. D., Mandrioli, D., McCullough, M.L., McNaughton, S.A., Morita, T., Nugent, A.P., Ogawa, K., Pandiri, A.R., Sergi, C.M., Touvier, M., Zhang, L., Benbrahim-Tallaa, L., Chittiboyina, S., Cuomo, D., DeBono, N.L., Debras, C., de Conti, A., El Ghissassi, F., Fontvielle, E., Harewood, R., Kaldor, J., Mattock, H., Pasqual, E., Rigutto, G., Simba, H., Suonio, E., Viegas, S., Wedekind, R., Schubauer-Berigan, M.K., Madia, F., 2023. Carcinogenicity of aspartame, methyleugenol, and isoeugenol. Lancet Oncol. 24, 848-850. https://doi.org/10.1016/S1470-2045(23)00341-8.

Salame, C., Javaux, G., Sellem, L., Viennois, E., de Edelenyi, F.S., Agaësse, C., De Sa, A., Huybrechts, I., Pierre, F., Coumoul, X., Julia, C., Kesse-Guyot, E., Allès, B., Fezeu, L. K., Hercberg, S., Deschasaux-Tanguy, M., Cosson, E., Tatulashvili, S., Chassaing, B., Srour, B., Touvier, M., 2024. Food additive emulsifiers and the risk of type 2 diabetes: analysis of data from the NutriNet-Santé prospective cohort study. Lancet Diabetes Endocrinol. 12, 339-349. https://doi.org/10.1016/S2213-8587(24)00086-X.

Schnabel, L., Buscail, C., Sabate, J.-M., Bouchoucha, M., Kesse-Guyot, E., Allès, B., Touvier, M., Monteiro, C.A., Hercberg, S., Benamouzig, R., Julia, C., 2018. Association between ultra-processed food consumption and functional gastrointestinal disorders: results from the French NutriNet-santé cohort. Am. J. Gastroenterol. 113, 1217-1228. https://doi.org/10.1038/s41395-018-01371-9.

Sellem, L., Srour, B., Javaux, G., Chazelas, E., Chassaing, B., Viennois, E., Debras, C., Salamé, C., Druesne-Pecollo, N., Esseddik, Y., de Edelenyi, F.S., Agaësse, C., De Sa, A., Lutchia, R., Louveau, E., Huybrechts, I., Pierre, F., Coumoul, X., Fezeu, L.K., Julia, C., Kesse-Guyot, E., Allès, B., Galan, P., Hercberg, S., Deschasaux-Tanguy, M., Touvier, M., 2023. Food additive emulsifiers and risk of cardiovascular disease in the NutriNet-Santé cohort: prospective cohort study. BMJ 382, e076058. https://doi.org/10.1136/bmj-2023-076058.

Srour, B., Chazelas, E., Druesne-Pecollo, N., Esseddik, Y., de Edelenyi, F.S., Agaësse, C., De Sa, A., Lutchia, R., Debras, C., Sellem, L., Huybrechts, I., Julia, C., Kesse-Guyot, E., Allès, B., Galan, P., Hercberg, S., Pierre, F., Deschasaux-Tanguy, M., Touvier, M., 2023. Dietary exposure to nitrites and nitrates in association with type 2
diabetes risk: results from the NutriNet-Santé population-based cohort study. PLoS Med. 20, e1004149. https://doi.org/10.1371/journal.pmed.1004149.

Srour, B., Chazelas, E., Fezeu, L.K., Javaux, G., Pierre, F., Huybrechts, I., Hercberg, S., Deschasaux-Tanguy, M., Kesse-Guyot, E., Touvier, M., 2022. Nitrites, Nitrates, and Cardiovascular Outcomes: Are We Living "La Vie en Rose" With Pink Processed Meats? J. Am. Heart Assoc. 11, e027627. https://doi.org/10.1161/JAHA.122.027627.

Srour, B., Fezeu, L.K., Kesse-Guyot, E., Allès, B., Debras, C., Druesne-Pecollo, N., Chazelas, E., Deschasaux, M., Hercberg, S., Galan, P., Monteiro, C.A., Julia, C., Touvier, M., 2020. Ultraprocessed food consumption and risk of type 2 diabetes among participants of the NutriNet-santé prospective cohort. JAMA Intern. Med. 180, 283-291. https://doi.org/10.1001/jamainternmed.2019.5942.

Srour, B., Fezeu, L.K., Kesse-Guyot, E., Allès, B., Méjean, C., Andrianasolo, R.M., Chazelas, E., Deschasaux, M., Hercberg, S., Galan, P., Monteiro, C.A., Julia, C., Touvier, M., 2019. Ultra-processed food intake and risk of cardiovascular disease: prospective cohort study (NutriNet-Santé). BMJ l1451. https://doi.org/10.1136/bmj.l1451.

Suez, J., Korem, T., Zeevi, D., Zilberman-Schapira, G., Thaiss, C.A., Maza, O., Israeli, D., Zmora, N., Gilad, S., Weinberger, A., Kuperman, Y., Harmelin, A., Kolodkin-Gal, I., Shapiro, H., Halpern, Z., Segal, E., Elinav, E., 2014. Artificial sweeteners induce glucose intolerance by altering the gut microbiota. Nature 514, 181-186. https://doi.org/10.1038/nature13793.

Touvier, M., Louzada, M.L. da C., Mozaffarian, D., Baker, P., Juul, F., Srour, B., 2023. Ultra-processed foods and cardiometabolic health: public health policies to reduce consumption cannot wait. BMJ 383, e075294. https://doi.org/10.1136/bmj-2023-075294.

Viennois, E., Merlin, D., Gewirtz, A.T., Chassaing, B., 2017. Dietary emulsifier-induced low-grade inflammation promotes colon carcinogenesis. Cancer Res. 77, 27-40. https://doi.org/10.1158/0008-5472.CAN-16-1359.

Yamada, M., Honma, M., 2018. Summarized data of genotoxicity tests for designated food additives in Japan. Gene Environ. 40, 27. https://doi.org/10.1186/s41021-018-0115-2.