Scientific Opinion on the re-evaluation of Brown FK (E 154) as a food additive

The Panel on Food Additives and Nutrient Sources added to Food provides a scientific opinion re-evaluating the safety of Brown FK (E 154). Brown FK is a food colouring substance permitted only in kippers at a maximum concentration of 20 mg/kg, which has been previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the EU Scientific Committee for Food (SCF) in 1983. The SCF established an Acceptable Daily Intake (ADI) of 0.15 mg/kg body weight (bw)/day. JECFA withdrew their temporary ADI of 0.075 mg/kg bw/day for Brown FK at their latest evaluation in 1987, as the toxicological data were considered inadequate. 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. The Panel calculated an anticipated refined dietary exposure to Brown FK for European children ranging from 0.03 to 0.07 mg/kg bw/day in average, and from 0.03 to 0.14 mg/kg bw/day at the 95th percentile. For the adult population the anticipated mean dietary exposure ranged from 0.002 to 0.07 mg/kg bw/day, and from 0.007 to 0.14 mg/kg bw/day at the 95th percentile. The Panel noted uncertainties surrounding the NOAELs reported for Brown FK in a chronic toxicity study and a carcinogenicity study in rats. It also noted the treatment-related changes occurring at the highest dose level in the latter study and that, as reported by JECFA, histopathological examination of the lower groups had not been carried out. The Panel considered, in line with JECFA, that this examination should have been carried out. The Panel considers therefore that it is unable to conclude on the safety of Brown FK given the deficiencies in the available toxicity database on the colour.

© European Food Safety Authority, 2010

Following a request from the European Commission to the European Food Safety Authority, the Scientific Panel on Food Additives and Nutrient Sources added to Food was asked to deliver a scientific opinion re-evaluating the safety of Brown FK (E 154) when used as a food colouring substance.

Brown FK (E 154) is a multi-component mixture of mono-, di- and tri-azo dyes authorised as a food additive in the EU and previously evaluated by both the EU Scientific Committee for Food (SCF) in 1984 and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1977, 1978, 1985, 1986 and 1987.

Brown FK is described as consisting of a mixture of sodium 4-(2,4-diaminophenylazo) benzenesulphonate (component I), sodium 4-(4,6-diamino-m-tolylazo)benzenesulphonate (component II), disodium 4,4’-(4,6-diamino-1,3-phenylenebisazo)dibenzenesulphonate (component III), disodium 4,4’-(2,4-diamino-1,3-phenylenebisazo)dibenzenesulphonate (component IV), disodium 4,4’-(2,4-diamino-5-methyl-1,3-phenylenebisazo)dibenzenesulphonate (component V) and trisodium 4,4’,4”-(2,4-diaminobenzene-1,3,5-trisazo)tribenzenesulphonate (component VI), together with subsidiary colouring matters and with water, sodium chloride and/or sodium sulphate as the principal uncoloured components.

The Panel noted that according to the CAS Registry Number, Brown FK is comprised of only components II and IV, rather than the six components mentioned by Commission Directive 2008/128/EC. The Panel also noted that, as reported by Emerton (2008), manufacture of Brown FK according to the EC specifications is extremely difficult and, given the limited usage of the colour and the availability of alternative colours, the major manufacturers have stopped offering the colour to EC specifications. The Panel noted therefore that there is some doubt about whether Brown FK is still used as a food colour in the European Union.

The SCF in its evaluation of Brown FK in 1984 established an Acceptable Daily Intake (ADI) of 0.15 mg/kg bw/day based on a No-Observed-Adverse-Effect-Level (NOAEL) of 15 mg/kg bw/day determined in a rat multi-generation study, while JECFA allocated a temporary ADI of 0.075 mg/kg bw/day for Brown FK based on a 2-year chronic toxicity study in rats. JECFA in its last evaluation in 1987 withdrew its temporary ADI due to the fact that additional data requested by the Committee at the time of establishing the temporary ADI, namely histopathological examination of the animals in the low and intermediate dose groups in a rat carcinogenicity study in order to establish a definitive NOAEL from the study, had not been provided.

The available studies on the absorption, distribution, metabolism and excretion of Brown FK indicate that the individual components of Brown FK are not absorbed as intact molecules to any extent as such, but undergo reductive cleavage in the intestine, with some subsequent absorption of aromatic polyamine metabolic products which are further acetylated before urinary excretion. Metabolites of Brown FK, including sulphanilic acid, are also excreted in the faeces. Overall given the multi-component nature of Brown FK, the metabolism of the mixture is complex but the absorption of metabolites of Brown FK, including aromatic amines, was demonstrated, as evidenced by the toxicity seen in specific studies with these metabolites.

Adverse effects seen after short-term or prolonged exposure to Brown FK in rats and/or mice include damage to cardiac (and to a minor extent skeletal) muscle fibres, characterised by vacuolar myopathy, degenerative lesions including myocardial fibrosis, necrosis and pigment deposition. Other changes induced by Brown FK include occasional hydropic degeneration of the kidney, fatty change of the liver, and increases in organ weight (spleen, liver, heart, testes and thyroid). The available studies provide no clear indication of a NOAEL for the cardiac toxicity or other pathological lesions seen following oral administration of Brown FK, other than that this NOAEL appears to lie below 100 mg/kg bw/day. In addition to the toxicity findings, pigmentation of tissues was seen from a dose of 55 mg/kg bw/day onwards in mice and 30 mg/kg bw/day onwards in rats. The lowest NOAEL reported from a dietary study in rats of 15 mg/kg bw/day was based on pigment deposition rather than biochemical or pathological indicators of toxicity.

In an in vitro bacterial mutagenicity study, different samples of Brown FK have been shown to induce concentration-dependent frame shift mutations (22- to 50-times the spontaneous mutation frequency) in Salmonella typhimurium (TA1538). The genotoxicity of samples of Brown FK has also been demonstrated in fluctuation assays in Salmonella typhimurium using TA1538 and in Escherichia coli WP2 uvrA, with and without rat liver S9 metabolic activation. The Panel noted that no information was available on the purity of the material tested in these studies and also noted the absence of in vitro and in vivo genotoxicity studies on the colour, other than bacterial mutagenicity assays. The Panel also noted that the different components of Brown FK are metabolised to non-sulphonated aromatic amines, and that potential impurities in the commercial product include m-phenylenediamine and 4-methyl-m-phenylenediamine and other non-specified non-sulphonated aromatic amines, the genotoxicity of which could be of concern. The Panel additionally noted that 4-methyl-m-phenylenediamine (toluene-2,4-diamine) has been assessed by the International Agency for Research into Cancer (IARC) as a Group 2B carcinogen (possibly carcinogenic to humans). While the Panel considered that the positive results obtained in in vitro bacterial mutagenicity studies on Brown FK, together with the potential genotoxicity of its metabolites and possible impurities, give rise to some concern regarding the genotoxicity of Brown FK, this concern is allayed by the results of long-term studies in rats and mice showing no convincing evidence of carcinogenicity of Brown FK.

Three long-term toxicological studies have been conducted, one in mice and two in rats. One of the rat studies was described as a 2-year chronic toxicity study, while the other rat study was a carcinogenicity study with in utero exposure followed by a 2-year exposure of the offspring. The mouse study was an 80-week carcinogenicity study. In the mouse study, hepatocellular carcinomas were observed, however the Panel considered that since the relevance of hepatic tumours in mice for humans has been questioned, the results of this study are unlikely to be of biological relevance and are therefore not a suitable basis for risk assessment. No indications for carcinogenicity were obtained in the long-term study with rats involving in utero exposure. The Panel concluded therefore that these long-term studies of Brown FK in rats and mice show no evidence of carcinogenic effects relevant to humans.

No evidence of reproductive toxicity of Brown FK was seen in a multi-generation study and a developmental toxicity study in rats.

The Panel noted that, in addition to the multi-generation study in rats, used by SCF to derive an ADI, either the 2-year chronic toxicity study in rats with a reported NOAEL of 15 mg Brown FK/kg bw/day (used by JECFA to derive their temporary ADI of 0.075 mg/kg bw/day for Brown FK) or the 2-year carcinogenicity study in rats involving an in utero exposure with a reported (by the authors) NOAEL of 50 mg/kg bw/day, might be considered to be appropriate studies to use as the basis for establishing an ADI.

The Panel did not however have access to the reports of any of these studies, and noted the uncertainties surrounding the NOAELs derived by the authors of the studies, including the relative contributions of treatment-related changes such as myopathy, myocardial fibrosis and pigment deposition in establishing effect and no-effect levels. In particular the Panel noted that according to JECFA, a range of treatment-related effects were reported in the 2-year carcinogenicity study at the highest dose of 250 mg/kg bw/day. However histopathological examination of the organs and tissues showing treatment-related changes in the highest dose group had not been carried out in the animals in the low and intermediate dose groups.

Since treatment-related effects had been observed at the top dose level of 250 mg/kg bw/day in this study, the Panel considered, in agreement with the view of JECFA, that histopathological examination of the relevant organs and tissues of (at least) the intermediate dose group animals should have been carried out. The Panel concluded that given the uncertainties in the available data sets, it was not possible to derive an appropriate NOAEL from the available data on these studies that could be used to establish an ADI. Therefore, the Panel is unable to conclude on the safety of Brown FK.

No data on sensitivity to Brown FK are available. Additionally, no cases of intolerance/allergenicity have been reported after oral exposure to Brown FK, and it appears therefore that at the current levels of exposure the incidence is very low, if any. On the other hand, the low/absent reports of adverse clinical reactions after internal Brown FK exposure could be accentuated by the lack of clinical awareness of this possibility.

Based on the fact that Brown FK is permitted only in kippers at a maximum concentration of 20 mg/kg, the Panel calculated an anticipated refined dietary exposure (Tier 2) for European children (aged 1-10 years and weighing 15-30 kg) ranging from 0.03 to 0.07 mg/kg bw/day in average, and from 0.03 to 0.14 mg/kg bw/day at the 95th percentile. For the adult population the anticipated mean dietary exposure ranged from 0.002 to 0.07 mg/kg bw/day, and from 0.007 to 0.14 mg/kg bw/day at the 95th percentile.

The Panel notes that the JECFA specification for lead in Brown FK is < 2 mg/kg, whereas the EC specification is < 10 mg/kg.

The Panel also notes that the aluminium lake of the colour could add to the daily intake of aluminium, for which a Tolerable Weekly Intake (TWI) 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.
 

Brown FK, E 154, CAS Registry Number 8062-14-4, food colouring substance, sodium 4-(2,4-diaminophenylazo)benzenesulphonate, sodium 4-(4,6-diamino-m-tolylazo)benzenesulphonate, disodium 4,4’-(4,6-diamino-1,3-phenylenebisazo)dibenzenesulphonate, disodium 4,