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

References

  1. FDA. Food and drug administration compliance program guidance manual. Chapter 03 —Foodborne biological hazards. USA: FDA; 2008. Available from: https://www.fda.gov/media/71245/download
  2. Agarwai K, Mukherjee A, Sharma A. In vivo cytogenetic studies on male mice exposed to Ponceau 4R and beta-carotene. Cytobios. 1993;74(296):23–28.
  3. Bateman B. The effects of a double blind, placebo controlled, artificial food colourings and benzoate preservative challenge on hyperactivity in a general population sample of preschool children. Archives of Disease in Childhood. 2004;89(6):506–511. doi: 10.1136/adc.2003.031435
  4. McCann D, Barrett A, Cooper A, et al. Food additives and hyperactive behaviour in 3-year-old and 8/9-year-old children in the community: a randomised, double-blinded, placebo-controlled trial. The Lancet. 2007;370(9598):1560–1567. doi: 10.1016/S0140-6736(07)61306-3
  5. EFSA Panel on Food Additives or Nutrient Soarces Added to Food. Scientific Opinion on the re-evaluation of Ponceau 4R (E 124) as a food additive. EFSA Journal. 2009;7(11):1328. doi: 10.2903/j.efsa.2009.1328
  6. Kirkland D, Reeve L, Gatehouse D, Vanparys P. A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2011;721(1):27–73. doi: 10.1016/j.mrgentox.2010.12.015 EDN: OKOVFF
  7. Yurchenko VV, Ingel FI, Akhaltseva LV, et al. Genotoxic safety of synthetic food colours. Review. Ecological genetics. 2021;19(4):323–341. doi: 10.17816/ecogen79399 EDN: MUULZS
  8. Ishidate M, Sofuni T, Yoshikawa K, et al. Primary mutagenicity screening of food additives currently used in Japan. Food and Chemical Toxicology. 1984;22(8):623–636. doi: 10.1016/0278-6915(84)90271-0
  9. Izbirak A, Sumer S, Diril N. Mutagenicity testing of some azo dyes used as food additives. Microbiyol Bul. 1990;24(1):48–56.
  10. Hayashi M, Matsui M, Ishii K, Kawasaki M. Data sheet for mutagenicity evaluation of food additives by Ministry of Health Labour and Welfare (FY1979-FY1998). Environ Mutagen Res. 2000;22:27–44.
  11. Haveiand-Smith RB. An evaiuation on the genetic effects of some food coiours using microbia test systems. Ph D. Thesis. London: CNAA; 1980.
  12. Yamjala K, Subramania Nainar M, Varma SK, Ambore N. Separation, identification and mutagenic assessment of the photodegradation products of Ponceau 4R (E124) in a beverage. Analytical Methods. 2016;8(25):5017–5024. doi: 10.1039/C6AY00716C
  13. Cameron TP, Hughes TJ, Kirby PE, et al. Mutagenic activity of 27 dyes and related chemicals in the Salmonella/microsome and mouse lymphoma TK+/− assays. Mutation Research/Genetic Toxicology. 1987;189(3):223–261. doi: 10.1016/0165-1218(87)90056-5
  14. Luck H, Rickerl E. Lebensmittelzusatzstoffe und mutagene Wirkung.VI.Pruefung der in Westdeutschland zugelassenen und urapruenglich vorgeachlagenen Lebensmittelfarbatoffe auf mutagene Wirkung an Escherichia coli. Z Lebens- mittel-Untersuch-Forsch. 1960;112:157–174. (In German)
  15. Sankaranarayanan N, Murthy MSS. Testing of some permitted food colours for the induction of gene conversion in diploid yeast. Mutation Research/Genetic Toxicology. 1979;67(4):309–314. doi: 10.1016/0165-1218(79)90026-0
  16. Gubbini L, Cardamone J, Voiterra-Veca L, et al. Controiio deii’ effetto mutageno di aicuni coioranti chimici ambientaii. Atti Ass. Genet Ital. 1975;20:43–44.
  17. Vaidya VG, Godbole NM. Mutagenicity stady of four colours using human leucocyte and mouse micronucleus test systems. Indian Journal of Experimental Biology. 1978;16(7):820–821.
  18. Yurchenko VV, Akhaltseva LV, Yurtseva NA, et al. Evaluation of mutagenic activity of the food dye Ponceau 4R in a micronuclear test in mice. Hygiene and sanitation. 2023;102(11):1210–1214. doi: 10.47470/0016-9900-2023-102-11-1210-1214 EDN: RIWBWA
  19. Durnev AD, Oreshchenko AV, Kulakova AV, Beresten NF. Analysis of cytogenetic activity of food dyes. Voprosy medicinskoj himii. 1995;41(5):50–53. EDN: UZFCRZ
  20. Bastaki M, Farrell T, Bhusari S, et al. Lack of genotoxicity in vivo for food color additive Tartrazine. Food and Chemical Toxicology. 2017;105:278–284. doi: 10.1016/j.fct.2017.04.034
  21. Sasaki YF, Kawaguchi S, Kamaya A, et al. The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2002;519(1-2):103–119. doi: 10.1016/s1383-5718(02)00128-6 EDN: AYFGGD
  22. Tsuda S. DNA Damage induced by red food dyes orally administered to pregnant and male mice. Toxicological Sciences. 2001;61(1):92–99. doi: 10.1093/toxsci/61.1.92 EDN: IWNCVD
  23. Yamada M, Honma M. Summarized data of genotoxicity tests for designated food additives in Japan. Genes and Environment. 2018;40(1):1–25. doi: 10.1186/s41021-018-0115-2 EDN: NMRVHY
  24. Shimada C, Kano K, Sasaki YF, et al. Differential colon DNA damage induced by azo food additives between rats and mice. The Journal of Toxicological Sciences. 2010;35(4):547–554. doi: 10.2131/jts.35.547
  25. Ingel FI. Part 2. Environmental factors and individual features in system of evaluation of human genome instability. additional capability of the test the technique for cytogenetic analysis. Ecological genetics. 2006;4(4):38–54. doi: 10.17816/ecogen4438-54 EDN: HZNVET
  26. Ingel FI, Yurchenko VV, Guskov AS, et al proliferative activity parameters and their correlation with genetic damage of blood lymphocytes duringultivation under the conditions of cytokinetic block. Annals of The Russian Academy of Medical Sciences. 2006(4):41–45. EDN: HSYNGB
  27. Swaroop VR, Roy DD, Vijayakumar T. Genotoxicity of synthetic food colorants. Journal of Food Science and Engineering. 2011;1:53–59.
  28. OECD. Test No. 487: In Vitro mammalian cell micronucleus test. OECD Guidelines for the Testing of Chemicals, Section 4. Paris: OECD; 2023. doi: 10.1787/9789264264861-en
  29. Yeropkin MYh, Yeropkina EM. Model of biotransfor mation of xenobiotics In vitro: effect of liver fraction S9 on toxicity of some an liviral preparations. Toxicological Review. 2008;(5):35–39. EDN: JVOOMT
  30. Ingel FI, Erdinger L, Eckl P, et al. Genomic instability, radiosensitivity and adaptive response of blood lymphocytes from children living in the aral sea region: correlation with emotional stress and blood contamination. Central European Journal of Occupational and Environmental Medicine. 2010;16(1-2):31–45. EDN: WTJSLP
  31. Ingel F, Krivtsova E, Urtseva N, et al. Volatility and sensitivity of the genome of healthy children in Magnitogorsk. Hygiene and Sanitation, Russian Journal. 2013;92(3):20–27. EDN: QIQPXV
  32. Nikitina TA, Konyashkina MA, Ingel FI, Akhaltseva LV. Evaluation of the genotoxic effect of tartrazine using a metabolic activation system in human lymphocyte culture under cytokinetic block conditions. Ecological Genetics. 2023;21(1):41–51. doi: 10.17816/ecogen117502 EDN: VADCQS
  33. Rastogi RP, Richa, Sinha RP. Apoptosis: molecular mechanisms and pathogenicity. EXCLI Journal. 2010;8:155–181. doi: 10.17877/DE290R-8930
  34. Veres IA. Apoptosis-dependent mechanisms of inflammation. Medical Journal. 2017;(3):147–152. EDN: ZEGMCP
  35. Lugovaya AV, Kalinina NM, Mitreikin VP, et al. Apoptosis and proliferation ofperipheral blood T-cells as alternative processes in pathogenesis of diabetes mellitus type 1. Medical alphabet. 2019;1(4):16–20. doi: 10.33667/2078-5631-2019-1-4(379)-16-20 EDN: PVXRKE
  36. Microbiological and molecular genetic assessment of food products obtained using genetically modified microorganisms: guidelines. Moscow: Federal Center for State Sanitary and Epidemiological Surveillance of the Ministry of Health of Russia; 2004. (In Russ.) Available from: https://meganorm.ru/Data2/1/4293855/4293855349.pdf

Supplementary files

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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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