Genotoxic effects of commercial sample of ponceau 4R-based food colorant in micronucleus assay on human whole blood culture

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Abstract

BACKGROUND: Genetic safety assessments of food additives have traditionally been conducted using highly purified substances. In the Russian Federation, according to the requirements of the Customs Union Technical Regulation, genotoxicity testing of approved food colorants is not mandatory. The regulation is limited to determining the content of the main colorant component and select constituents. However, this approach is insufficient, as it does not account for the possible presence of toxic and genotoxic impurities in food colorants.

AIM: To evaluate the genetic safety of a commercially available Ponceau 4R (E124)-based food colorant using the micronucleus assay on human whole blood culture with cytokinesis block, both in the presence and absence of a metabolic activation system.

METHODS: The Ponceau 4R-based colorant was purchased in a retail store. The cells from a healthy donor were cultured under cytokinesis-block conditions with and without S9 rat liver metabolic activation, exposed to the colorant at concentrations ranging from 0 to 2 mg/mL. Cytome assay was performed using an extended micronucleus assay protocol. Statistical analysis was conducted using the χ2 test and the Mann–Whitney U test.

RESULTS: A statistically significant increase in the frequency of cells with genetic damage was observed, following a U-shaped dose–response pattern. Without metabolic activation, significant effects were found at concentrations of 0.0000256, 0.00064, and 0.4 mg/mL; with S9 activation, at concentrations of 0.0000256, 0.000128, and 0.016 mg/mL. In addition, in the presence of the S9 fraction, an increased frequency of trinucleated cells, stimulation of mitotic activity, and suppression of apoptosis were also observed.

CONCLUSION: Genotoxic effects of the Ponceau 4R-based food colorant obtained from the retail market were detected at or below the acceptable daily intake level for humans. The proposed approach may serve as a foundation for the development of a system for genetic safety assessment of food colorants and additives.

About the authors

Tatyana A. Nikitina

Centre for Strategic Planning and Management of Biomedical Health Risks

Author for correspondence.
Email: TNikitina@cspmz.ru
ORCID iD: 0000-0003-0866-5990
SPIN-code: 9106-5076
Russian Federation, Moscow

Maria A. Konyashkina

Centre for Strategic Planning and Management of Biomedical Health Risks

Email: MKonyashkina@cspfmba.ru
ORCID iD: 0000-0002-8319-1329
SPIN-code: 7559-9045
Scopus Author ID: 8142882800

Cand. Sci. (Biology)

Russian Federation, Moscow

Faina I. Ingel

Centre for Strategic Planning and Management of Biomedical Health Risks

Email: FIngel@cspmz.ru
ORCID iD: 0000-0002-2262-6800
SPIN-code: 1013-7006
Scopus Author ID: 57205760994
ResearcherId: C-8899-2014

Dr. Sci. (Biology)

Russian Federation, Moscow

Lyudmila V. Akhaltseva

Centre for Strategic Planning and Management of Biomedical Health Risks

Email: LAhalceva@cspmz.ru
ORCID iD: 0000-0002-3619-3858
SPIN-code: 7049-0003
Scopus Author ID: 57138478700
ResearcherId: I-8204-2018

Cand. Sci. (Biology)

Russian Federation, Moscow

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Supplementary files

Supplementary Files
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1. JATS XML
2. Fig. 1. Control cultures: spectrum of cells that completed varying numbers of division cycles in the presence of cytochalasin B. S9, metabolic activation system.

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3. Fig. 2. Effect of food coloring on cell proliferation: a – effect of Ponceau 4R only; b – effect of Ponceau 4R under conditions of metabolic activation.

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4. Fig. 3. Pairwise comparison of cell population spectra in human blood cultures after exposure to the food colorant with and without metabolic activation: red star, statistically significant differences in the frequency of trinucleated cells within the spectrum of cell populations under different cultivation conditions; arrows, statistically significant differences in the frequency of cells that completed the same number of division cycles under different cultivation conditions (the direction indicates the vector of change, and the color corresponds to the cell fraction for which the difference is statistically significant). S9, metabolic activation system.

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