Scientific Opinion on the re-evaluation of Brown HT (E 155) 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 HT (E 155). Brown HT has been previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1977 and 1984 and the EU Scientific Committee for Food (SCF) in 1975 and 1984. JECFA established an Acceptable Daily Intake (ADI) of 0-1.5 mg/kg body weight (bw)/day, while the SCF established an ADI of 0-3 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. The Panel concluded that based on a long-term carcinogenicity and toxicity study in mice an ADI of 1.5 mg/kg bw/day can be derived. The Panel concludes that at both the maximum permitted level of use (Tier 2) and at the maximum reported levels of use of Brown HT (Tier 3), intake estimates are generally below the ADI of 1.5 mg/kg bw/day. However, in both adults and 1-10 years old children, the high percentile of exposure for both Tiers can be slightly higher than the ADI at the upper end of the range.

© European Food Safety Authority, 2010

Following a request from the European Commission to the European Food Safety Authority (EFSA), the Scientific Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to provide a scientific opinion re-evaluating the safety of Brown HT (E 155) when used as a food colouring substance.

Brown HT (E 155) is a synthetic bis-azo dye authorised as a food additive in the EU and previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1977 and the EU Scientific Committee for Food (SCF) in 1984. In 1984 JECFA established an Acceptable Daily Intake (ADI) of 0-1.5 mg/kg bw/day, while the SCF established an ADI of 0-3 mg/kg bw/day.

Based on studies with radioactive Brown HT, the Panel concluded that Brown HT or its metabolites are absorbed to a limited extent in mice, rats and guinea pigs and are excreted predominantly in faeces (up to 90%) and urine (13-16%). Faecal extracts of mice and rats contained only small amounts of unchanged Brown HT, naphthionic acid and two unidentified metabolites. Urine contained naphthionic acid and one unidentified metabolite. These findings indicate that the azo-bonds of Brown HT are reductively cleaved by intestinal bacteria as is the case with other azo-dyes. It is unclear if the central ring structure (2,4-dihydroxy-3,5-diamino benzyl alcohol; an aromatic amine) is released. The radiolabel was distributed limitedly and mostly associated with the gastrointestinal tract and to a minor extent with the liver and kidney in both rats and male mice. Brown HT and/or its metabolites deposited only in the kidney and mesenteric lymph nodes.

In a short-term toxicity studies of Brown HT in rats, effects generally appeared compound-related but not dose-related, or were observed at doses that would not lead to a lowering of the current ADI.

Bacterial genotoxicity tests with Brown HT have been negative. Because the activation process of these bis-azo dyes in animals is complex, bacterial tests with S9 might not be suitable to detect mammalian genotoxicity.

The Panel noted that Prival and Mitchell (1982) demonstrated that the metabolic conditions of the standard Ames test protocol were not appropriate for testing azo dyes for mutagenic activity in Salmonella typhimurium and developed a specific protocol including use of flavin mononucleotide (FMN) rather than riboflavin to reduce the azo compounds to free amines, and hamster liver S9 rather than rat liver S9 for metabolic activation. The Panel therefore noted that a final conclusion from negative Ames test results obtained under standard conditions cannot be drawn. The conversion of the parent compound by azo-reduction in vivo results in the formation of sulphonated naphthylamines as well as unsulphonated aromatic amines that may not be formed in the standard in vitro genotoxicity tests. Previously, a range of sulphonated aromatic amines, including the ones formed from Brown HT upon azo-reduction such as naphthionic acid, was shown to be in general not associated with genotoxicity in vitro and in vivo. In contrast with their unsulphonated analogues they have no or very low genotoxic potential. Hence it was concluded that exposure to sulphonated aromatic amines present in colourings, are unlikely to induce any significant genotoxic risk.

The Panel also noted that the specifications on the purity of Brown HT permit concentrations of unidentified unsulphonated aromatic amines to be present in concentrations of up to 100 mg/kg Brown HT. Although some aromatic amines may be associated with genotoxicity or even carcinogenicity, the Panel notes that Brown HT was negative in in vitro genotoxicity as well as in long-term carcinogenicity studies.

Long term toxicity and carcinogenicity studies with Brown HT are available with rats and mice. No carcinogenic effects were observed in either species. No adverse effects were reported in rats at dietary dose levels up to 425 mg/kg bw/day (highest dose tested). Several effects were observed in the long-term mouse study at the highest dose tested (715 mg 85% pure Brown HT/kg bw/day). The NOAEL in the mouse study was 0.1% in the diet equivalent to 143 mg Brown HT/kg bw/day.

The negative outcome of the carcinogenicity studies is considered by the Panel to rule out the concern on potential genotoxicity of the unsulphonated aromatic amine which may result from azoreduction of Brown HT by intestinal bacteria. The Panel considered that in the light of the results of the available carcinogenicity studies there is no need for additional genotoxicity studies.

Conceivably the same study was used by SCF and JECFA for calculation of the ADI. No further details on both the SCF and JECFA evaluations are available.

The Panel notes that no cases of intolerance/allergenicity/hypersensitivity have been reported after oral exposure to Brown HT and that no data on sensitivity to Brown HT are available.

The ADI of 0-1.5 mg/kg bw/day defined by JECFA was based on a NOAEL of 143 mg/kg bw/day derived from the long-term feeding study in mice. Although it appears that both JECFA and SCF have derived a NOAEL from the same study, their respective ADIs for 85% pure Brown HT have been set at 0-1.5 and 0-3 mg/kg bw/day respectively. No further details on both the SCF and JECFA evaluations are available.

Brown HT was tested for reproduction toxicity in a dietary three-generation study in rats revealing a NOAEL of 250 mg Brown HT/kg bw/day. In a developmental toxicity study in rats no teratogenic and embryotoxic effects were observed up to a dose level of 1000 mg/kg bw/day.

The Panel noted that although no new studies are available since previous evaluations, at present these old studies have been reviewed and published in scientific journals.

The Panel concluded that an ADI of 1.5 mg Brown HT/kg bw/day can be established based on the NOAEL in a long-term mouse study of 143 mg/kg bw/day and an uncertainty factor of 100 and rounding off the ADI of 1.5 mg/kg bw/day.

The dietary exposure to Brown HT 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 for adults, and 8.1 mg/kg bw/day for a typical 3 year-old child.

Refined exposure estimates have been performed both for children 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 the Directive 94/36/EC on food colours (Tier 2), and with the maximum reported use levels of Brown HT listed in Table 3 (Tier 3), as identified by the Panel from the data made available by the FSA and UNESDA For children (1-10 years old), estimates have been calculated for nine European countries (Belgium, France, the Netherlands, Spain, Italy, Finland, Greece, Sweden and Germany) and for UK children separately. For the adult population, the Panel has selected the UK population as representative of the EU consumers for Brown HT exposure estimates.

When considering MPLs (Tier 2), the mean dietary exposure to Brown HT for European children, (aged 1-10 years), ranged from 0.3 to 2.2 mg/kg bw/day, and from 0.8 to 5.9 mg/kg bw/day at the 95th percentile. Estimates reported for the UK adult population give a mean dietary exposure of 0.5 mg/kg bw/day and of 2.9 mg/kg bw/day for high level (97.5th percentile) consumers of ‘aromatized fruit wines, cider and perry’.

When considering the maximum reported use levels (Tier 3), the mean dietary exposure to Brown HT for European children (aged 1-10 years) ranged from 0.3 to 2.0 mg/kg bw/day, and from 0.7 to 5.8 mg/kg bw/day at the 95th percentile. Estimates reported for the UK adult population give a mean dietary exposure to Brown HT of 0.4 mg/kg bw/day, and of 2.8 mg/kg bw/day for high level (97.5th percentile) consumers of soft drinks.

The Panel concludes that at both the maximum permitted level of use (Tier 2) and at the maximum reported levels of use of Brown HT (Tier 3), mean intake estimates are generally below the ADI of 1.5 mg/kg bw/day. However, in both adults and children, the high percentile of exposure (97.5th) for both tiers can be higher than the ADI established by the Panel.

The Panel further notes that the specifications for Brown HT need to be updated with respect to the percentage of material not accounted. Thus, if the existing specifications would be extended to include < 30% of sodium or calcium chloride and/or sodium or calcium sulphate as the principal uncoloured components, most of the material would be accounted for.

The Panel noted that the JECFA specification for lead is ≤ 2 mg/kg whereas the EC specification is ≤ 10 mg/kg.

The Panel noted that the aluminium lake of the colour could add to the daily intake of aluminium for which a TWI of 1 mg aluminium/kg bw/week has been established (EFSA, 2008) and that therefore specifications for the maximum level of aluminium in the lakes may be required.

Brown HT, E 155, CAS Registry Number 4553-89-3, disodium 4,4’-(2,4-dihydroxy-5-hydroxymethyl-1,3-phenylene bis-azo) di-(naphthalene-1-sulfonate), synthetic food colorant