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Scientific Opinion on the re-evaluation of Ponceau 4R (E 124) as a food additive
The Panel on Food Additives and Nutrient Sources added to Food provides a scientific opinion re-evaluating the safety of Ponceau 4R (E 124). Ponceau 4R has been previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1983 and the EU Scientific Committee for Food (SCF) in 1984. Both
committees established an Acceptable Daily Intake (ADI) of 0-4 mg/kg bw/day. The Panel was not provided with a newly submitted dossier and based its evaluation on previous evaluations, additional literature that became available since then and the data available following a public call for data. Relevant new studies included a study by Tsuda et al. from 2001 reporting effects on nuclear DNA migration in the mouse in vivo Comet assay, a study by Tanaka from 2006 on neurobehavioural effects and a study by McCann et al. from 2007 that concluded that exposure to a mixture including Ponceau 4R resulted in increased hyperactivity in 3-year old children. The Panel notes that Ponceau 4R was negative in in vitro genotoxicity as well as in long term carcinogenicity studies and that the effects on nuclear DNA migration are not expected to result in carcinogenicity. The Panel also concurs with the conclusion from a previous EFSA opinion on the McCann et al. study that the findings of the study cannot be used as a basis for altering the ADI. The Panel also re-evaluated a long-term mouse study reporting glomerulonephrosis from which they derived a No-Observed-Adverse-Effect Level of 70 mg/kg bw/day. Based on these findings the Panel derives an ADI of 0.7 mg/kg bw/day. The Panel concludes that at the maximum levels of use, intake estimates for adults at the high percentile (97.5th) and for 1- to 10-year old children at the mean and the high percentiles (95th/97.5th) are generally above the ADI even in the refined intake estimates.
© European Food Safety Authority, 2009
Following a request from the European Commission to the European Food Safety Authority (EFSA), the Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion re-evaluating the safety of Ponceau 4R (E 124) when used as a food colouring substance.
Ponceau 4R (E 124) is an azo dye allowed as a food additive in the EU that has been previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1983 and the EU Scientific Committee for Food (SCF) in 1984. Both committees established an Acceptable Daily Intake (ADI) of 0-4 mg/kg body weight (bw)/day.
Recent results indicated that in an in vivo Comet assay, Ponceau 4R induced significant increases in migration of nuclear DNA in glandular stomach, bladder and colon tissue, in the absence of general cytotoxicity in these tissues. The Panel considered in the light of negative carcinogenicity studies, that the biological significance of the Comet assay results is uncertain.
In contrast to this, all Salmonella genotoxicity tests with Ponceau 4R have been negative. Because the activation process of these azo dyes in animals is complex, Salmonella tests with S9 might not be suitable to detect mammalian genotoxicity.
The conversion of Ponceau 4R by azo reduction in vivo results in the formation of sulphonated naphthylamines that may not be formed in the standard in vitro genotoxicity tests. Previously, a range of sulphonated aromatic amines was shown to be in general not associated with genotoxicity in vitro and in vivo. However, not all the sulphonated aromatic amine metabolites that could in theory be formed by azo reduction of Ponceau 4R were included in the study.
The Panel also noted that the specifications on the purity of Ponceau 4R permit concentrations of unidentified unsulphonated aromatic amines to be present in concentrations of up to 100 mg/kg Ponceau 4R. Although some aromatic amines may be associated with genotoxicity or even carcinogenicity, the Panel noted that Ponceau 4R was negative in long term carcinogenicity studies.
Long-term carcinogenicity studies on Ponceau 4R were re-evaluated by the Panel. Several long-term carcinogenicity studies in rats at dose levels up to 1500 mg/kg bw/day, and in mice at dose levels up to 1790 mg/kg bw/day, revealed no evidence of carcinogenicity. This included the absence of neoplasms in the stomach or blood forming tissues, shown to be sensitive organs in the in vivo Comet assay in mice. Ponceau 4R induced significant dose-related DNA damage in mice in the glandular stomach and bladder at doses of 100 mg/kg bw and higher, and in the colon at doses of 10 mg/kg bw and higher. In bone marrow cells of male mice, clastogenic activity was noted at a minimum effective dose of 4 mg Ponceau 4R, equivalent to a dose of 80 mg/kg bw. However, carcinogenicity was not observed at dose levels several times higher, up to 1790 mg/kg bw/day for mice, and up to 1500 mg/kg bw/day in rats. The Panel noted that Ponceau 4R was negative in long term carcinogenicity studies and that the effects on nuclear DNA migration observed in the mouse in vivo Comet assay are not expected to result in carcinogenicity.
Based on the same dataset for long-term toxicity/carcinogenicity, previous evaluations by JECFA, the SCF and TemaNord also concluded that there was no evidence for carcinogenicity of Ponceau 4R.
A study by McCann et al. has concluded that upon exposure to two mixtures of four synthetic colours, plus the preservative sodium benzoate in the diet, one of them, Mix A (containing Ponceau 4R) resulted in increased hyperactivity in 3-year old, but not in 8- to 9-year old children in the general population. In 2008, EFSA also published an opinion on this McCann et al. study.
The Scientific Panel on Food Additives, Flavourings, Processing Aids and Food Contact Materials (AFC) concluded that:
- the McCann et al. study provides limited evidence that the two different mixtures of synthetic colours and sodium benzoate tested had a small and statistically significant effect on the activity and attention in children selected from the general population, excluding children medicated for Attention Deficit Hypersensitivity Disorder, although the effects were not statistically significant for the two mixtures in both age groups;
- since mixtures, and not individual additives, were tested in the study by McCann et al., it is not possible to ascribe the observed effects to any of the individual compounds, and;
- in the context of the overall weight of evidence and in view of the considerable uncertainties, such as the lack of consistency and relative weakness of the effect and the absence of information on the clinical significance of the behavioural changes observed, the findings of the study cannot be used as a basis for altering the ADI of the respective food colours or sodium benzoate.
The Scientific Panel on Food Additives and Nutrient Sources added to Food concurs with these conclusions.
A rat study by Tanaka, reported in 2006, concluded that the No Observed Adverse Effect Level (NOAEL) was presumed to be 0.12% in the diet (approximately 205 mg/kg bw/day) for maze learning by males in the F1 generation. The Panel notes that these neurobehavioural findings were not consistent among sexes and were especially observed because of reduced values in the control group.
The Panel noted that also two studies already available at the time JECFA and the SCF set the ADI, point at NOAEL values lower than 400 mg/kg bw/day. These include:
- a pig study from Gaunt et al. from 1969 reporting a NOAEL of 300 mg/kg bw/day based on a slight reduction in the number of erythrocytes at 900 mg/kg bw/day, and,
- the mouse study of Mason et al. from 1974 that concluded that the no-effect level, based on the findings of glomerulonephrosis at the 0.25 and 1.25% dietary levels, was 0.05%, equivalent to 70 mg/kg bw/day.
Overall, the Panel concluded that these findings do give reason for re-definition of the ADI. Based on the lowest NOAEL of 70 mg/kg bw/day from the long term mouse study and an uncertainty factor of 100, the Panel derives an ADI of 0.7 mg/kg bw/day.
The Panel concluded that while some sensitivity reactions after Ponceau 4R intake have been reported, mostly when Ponceau 4R is taken within mixtures of other synthetic colours, no conclusion on the induction of sensitivity by Ponceau 4R could be drawn from the limited scientific evidence available. The Panel also noted that sensitive individuals may react at dose levels within the ADI.
The dietary exposure to Ponceau 4R was estimated by the Panel based on the Maximum Permitted Levels (MPLs) of use, by applying the Budget method (Tier 1) with the assumptions described in the report of the Scientific Cooperation (SCOOP) Task 4.2. The Panel calculated a theoretical maximum daily exposure of 8.1 mg/kg bw/day both for adults and for a typical 3 year-old child.
Refined exposure estimates have been performed both forchildren and the adult population according to the Tier 2 and Tier 3 approaches described in the SCOOP Task 4.2, which combines, respectively, detailed individual food consumption information from the population with the MPLs of use as specified in Directive 94/36/EC on food colours (Tier 2) and with the maximum reported use levels, as identified by the Panel from the data made available by the UK Food Standards Agency, the Food Safety Authority of Ireland, the Agence Française de Sécurité Sanitaire des Aliments, the Union of European Beverage Associations, the European Spirits Organisation, the Federation of European Food Additives, Food Enzymes and Food Culture Industries, the Confederation of the Food and Drink Industries of the EU (Tier 3). For children (aged 1-10 years), estimates have been calculated for nine European countries (Belgium, France, the Netherlands, Spain, UK, Czech Republic, Italy, Finland, Germany). For the adult population, the Panel has selected the UK population as representative of the EU consumers for Ponceau 4R intake estimates.
When considering MPLs (Tier 2), the mean dietary exposure to Ponceau 4R for European children (aged 1-10 years) ranged from 0.3 to 2.5 mg/kg bw/day and from 0.6 to 6.7 mg/kg bw/day at the 95th percentile. Estimates reported for the UK adult population give a mean dietary exposure to Ponceau 4R of 0.5 mg/kg bw/day and of 1.1 mg/kg bw/day for high level (97.5th percentile) consumers of soft drinks.
When considering the maximum reported use levels (Tier 3), the mean dietary exposure of European children (aged 1-10 years) ranged from 0.3 to 2.4 mg/kg bw/day and from 0.7 to 6.2 mg/kg bw/day at the 95th percentile. Estimates reported for the UK adult population give a mean dietary exposure of 0.4 mg/kg bw/day and of 1.0 mg/kg bw/day for high level (97.5th percentile) consumers of soft drinks.
The Panel concludes that at the maximum levels of use of Ponceau 4R, intake estimates for adults at the high percentile (97.5th) and for 1- to 10-year old children at the mean and the high percentiles (95th/97.5th) are generally above the ADI of 0.7 mg/kg bw/day even in the refined intake estimates (Tier 2 and Tier 3).
The Panel further notes that the specifications for Ponceau 4R need to be updated with respect to the percentage of material not accounted for that may represent sodium chloride and/or sodium sulphate as the principal uncoloured components.
The Panel notes that the JECFA specification for lead is ≤ 2 mg/kg whereas the EC specification is ≤ 10 mg/kg.
The Panel notes that the aluminium lake of the colour could add to the daily intake of aluminium for which a Tolerable Weekly Intake of 1 mg aluminium/kg bw/week has been established and that therefore specifications for the maximum level of aluminium in the lakes may be required.
Ponceau 4R, New Coccine, E 124, CAS 2611-82-7, Trisodium 2-hydroxy-1-(4-Sulphonato-1-naphthylazo)- naphtalene-6,8-disulphonate, food colouring substance, EINECS number: 220-036-2.