The present opinion deals with the re‐evaluation of the safety of food‐grade carrageenan (E 407) and processed Eucheuma seaweed (E 407a) used as food additives.
Carrageenan (E 407) and processed Eucheuma seaweed (E 407a) are authorised as food additives in the European Union (EU) in accordance with Annex II and Annex III to Regulation (EC) No 1333/2008 on food additives and specific purity criteria have been defined in the Commission Regulation (EU) No 231/20121. In the EU, these two food additives have been evaluated by the Scientific Committee for Food (SCF) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in different occasions. The SCF (1978), endorsed the acceptable daily intake (ADI) of 0–75 mg/kg body weight (bw) per day for carrageenan (E 407) previously established by JECFA (1974). JECFA in 1984 changed to an ADI ‘not specified’ (JECFA, 1984), and in 2002, a group ADI ‘not specified’ was allocated to the sum of carrageenan (E 407) and processed Eucheuma seaweed (E 407a) (JECFA, 2002). The SCF maintained the original group ADI for all types of carrageenan (E 407) and processed Eucheuma seaweed (E 407a) of 0–75 mg/kg bw because of some uncertainty ‘about the general immuno‐reactive potential of the various carrageenans now in use as food additives’ (SCF, 1996). In 2003, the SCF had no objection to the use of carrageenan in follow‐on formulae up to a maximum level of 0.3 g/L (SCF, 2003b). In its latest evaluation, JECFA in 2015, concluded that ‘the use of carrageenan in infant formula or formula for special medical purposes at concentrations up to 1,000 mg/L is not of concern’ (JECFA, 2015).
According to the Commission Regulation (EU) No 231/2012, ‘carrageenan (E 407) consists chiefly of the potassium, sodium, magnesium and calcium sulphate esters of galactose and 3,6‐anhydrogalactose polysaccharides. The prevalent polysaccharides in carrageenan are designated as κ‐, ι‐, λ‐ depending on the number of sulphate by repeating unit (i.e. 1,2,3 sulphate)’. In the processed Eucheuma seaweed (E 407a), the main polysaccharide is κ‐carrageenan.
Because of the structural similarities of processed Eucheuma seaweed and the conventionally processed food‐grade carrageenan and the similarities of effects they caused in the comparative study (Documentation provided to EFSA n. 38), the Panel concluded that the re‐evaluation of processed Eucheuma seaweed (E 407a) can be included in that of food‐grade carrageenan (E 407).
According to one of the interested parties (Documentation provided to EFSA n. 19), carrageenan has a molecular weight distribution from 30 kDa to as high as 5,000 kDa and is defined as having a weight‐average molecular weight between 200 and 800 kDa. Furthermore, it has been confirmed by industry (Documentation provided to EFSA n. 19) that commercial carrageenan (E 407) may have a weight‐average molecular weight as low as 200 kDa. In view of the Panel, the molecular weight distribution of such a carrageenan product may have a considerable fraction of molecules encompassing weight‐average molecular weight of degraded carrageenan.
The Panel noted that in the literature the information on the molecular weight in the individual carrageenan preparation is often unprecise mixing the terms ‘molecular weight’, ‘weight‐average molecular weight’ and ‘number‐average molecular weight’.
The debate in literature with respect to the safety of native (undegraded) carrageenan has been related to the presence of degraded carrageenan. Degraded carrageenan is also known in forms of artificially produced products from carrageenan (e.g. poligeenan, C16) associated with adverse effects. Poligeenan is a much lower weight‐average molecular weight polymer (10–20 kDa) generated by subjecting ι‐carrageenan to the extreme conditions of acid hydrolysis at low pH (0.9–1.3) and high temperatures (> 80°C) for several hours (Cohen and Ito, 2006; McKim, 2014; JECFA, 2015; USDA, 2016). C16 is artificially formed from ι‐carrageenan under conditions of acidic hydrolysis with 0.1 M sulfuric acid, at 60°C for 1.5 h (Glaxo Lab).
The Panel emphasised that degraded carrageenan (e.g. poligeenan or C16) has not been authorised as a food additive in the EU. Information available from the US indicated that poligeenan is not used in any food applications (USDA, 2016). The Panel further noted that the term ‘poligeenan’ has been used in literature including test protocols in a broader sense. However, more specifically poligeenan is a breakdown product of ι‐carrageenan consisting of the same sulfated galactose and sulfated anydrogalactose units, linked by α‐1,3‐ and β‐1,4‐glycosidic bonds as in all types of carrageenan. According to information provided from interested parties (Documentation provided to EFSA n. 18), poligeenan comprises polysaccharide molecules of molecular weight ranging from approximately 2 to 200 kDa, and the weight‐average molecular weight of poligeenan ranges from 10 to 20 kDa. The Panel noted that based on the data provided (Documentation provided to EFSA n. 18) the overlapping between the low molecular weight tail of carrageenan and high molecular weight tail of poligeenan is increasing with the decrease of the weight‐average molecular weight of the carrageenan preparation.
Uno et al. (2001a), in a survey of 29 samples of food‐grade carrageenan representing κ‐, ι‐ and λ‐carrageenan determined a number‐average molecular weight of 193–324 kDa and a weight‐average molecular weight of 453–652 kDa. According to Uno et al. (2001a), ‘no obvious peak of poligeenan was detected’ and the ‘detection limit’ for poligeenan was about 5% in the sample of carrageenan.
According to information from one of the interested parties (Documentation provided to EFSA n. 19), ‘in native carrageenan, this low molecular weight tail (LMT) region represents less than 10% of the total carrageenan molecule. Although there is a Commission specification of not more than 5% at less than 50 kDa, in practice there is no validated analytical method that can accurately quantify the percent of LMT present’. The Panel noted that, according to the interested parties, an interlaboratory validated analytical method to accurately measure the low molecular weight distributions of carrageenan is not fully developed or available at present.
The Panel noted that in most toxicity studies no adequate information on the molecular weight distribution of carrageenan was available. Therefore, it was not possible to assess whether the carrageenan tested complied with the EU specifications (specially the limit of low molecular weight carrageenan < 5% below 50 kDa). Furthermore, the Panel noted the methodological limitation of in vivo studies, in which carrageenan was given in drinking water, which is not representative of the dietary exposure where food‐grade carrageenan will be bound to protein. Therefore, the extent to which drinking water studies might be representative for food‐grade carrageenan used as a food additive and consequently of the relevance of these studies for its safety assessment was deemed limited.
The available database on processed Eucheuma seaweed was limited to the endpoints of subchronic toxicity and genotoxicity but was sufficient for the evaluation of the similarities between processed Eucheuma seaweed and food‐grade carrageenan.
Carrageenan (E 407) and processed Eucheuma seaweed (E 407a) are authorised in a wide range of foods. The Panel did identify brand loyalty for specific food categories, e.g. flavoured fermented milk products for infants and toddlers, thus the Panel considered this scenario as the most appropriate and realistic scenario for risk characterisation for all population groups.
A maximum estimated exposure assessment scenario taking into account the food for special medical purposes for infants and young children (FC 18.104.22.168. Dietary foods for infants for special medical purposes and special formulae for infants, and FC 22.214.171.124. Dietary foods for babies and young children for special medical purposes as defined by Commission Directive 1999/22/EC) was also performed using food industry data to estimate exposure for infants and toddlers who may be on a specific diet. This exposure scenario considered products belonging to food categories 13.1.2 and 13.1.4 excluding food category 13.1.1 (infant formulae) and 13.1.3 (processed cereal‐based foods) where carrageenan (E 407) and processed Eucheuma seaweed (E 407a), according to EU Regulation, are not authorised. Considering that this diet is required due to specific needs, it is assumed that consumers are loyal to the food brand, therefore, the maximum reported use level for the foods for special medical purposes (FSMP) was used and the mean of the typical reported for the remaining food categories.
The refined estimates were based on 41 out of 79 food categories in which carrageenan (E 407) and processed Eucheuma seaweed (E 407a) are authorised. Overall, the Panel considered that the uncertainties identified would, in general, result in an overestimation of the real exposure to carrageenan (E 407) and processed Eucheuma seaweed (E 407a) as food additives in European countries for both the maximum level exposure scenario and for the refined exposure assessment scenarios when considering only food additives uses for which data have been provided.
No information on the usage levels of carrageenan (E 407) and processed Eucheuma seaweed (E 407a) has been reported to EFSA for 19 food categories. However, the Panel noted that, given the information from the Mintel Global New Products Database (GNPD), it may be assumed that carrageenan (E 407) and processed Eucheuma seaweed (E 407a) are used in several food categories (n = 7) for which no data have been provided by food industry. The Panel noted that out of these 19 food categories, pasta and breakfast cereals are products highly consumed. If this would be confirmed, it would therefore result in an underestimation of the exposure.
For FSMP, the Panel noted that one industry reported a use of carrageenan as food additive at the level of the maximum permissible level (MPL) (300 mg/kg), according to the Annex II to Regulation (EC) No 1333/2008, in one niche product which is not align with information reported from the Mintel GNPD (Appendix D), where no FSMP products seem to be labelled on the market. If this information is confirmed, the uncertainties arising from the FSMP exposure scenario would lead to an overestimation of the current exposure to carrageenan products as food additives calculated by the Panel.
Concerning uses of carrageenan in food for infants and young children the Panel concurred with the SCF (1998a, 2003a,b) ‘In the absence of any further information on possible absorption of carrageenan by the immature gut in the very young infant, the Committee reaffirms its earlier view (SCF, 1998a) that it remains inadvisable to use carrageenan in infant formulae that are fed from birth, including those in the category of foods for special medical purposes. The Committee has no objection to the use of carrageenan in foods for older infants, such as follow‐on milks (SCF, 1983) and weaning foods (SCF, 2003a)’.
In its evaluation of carrageenan (E 407) and processed Eucheuma seaweed (E 407a), the Panel noted that:
- according to industry carrageenan (E 407) is defined as having a weight‐average molecular weight of 200–800 kDa;
- Uno et al. (2001a) determined in a survey on samples of food‐grade carrageenan, representing κ‐, ι‐ and λ‐carrageenan, a weight‐average molecular weight of 453–652 kDa and detected no obvious peak of poligeenan with a detection limit for poligeenan of about 5%;
- the ADME database was sufficient to conclude that high molecular weight carrageenan (e.g. κ/λ‐carrageenan with a number‐average molecular weight of approx. 800 kDa, study in rhesus monkeys) was not absorbed intact, while low molecular weight carrageenan (number‐average molecular weight: 88 kDa or less) was found in tissues of rats or guinea pigs after administration of this material by gavage or diet;
- in one subchronic toxicity good laboratory practice (GLP) study in rats performed with κ‐carrageenan with an average molecular weight range of 196–257 kDa (not specified if number‐ or weight‐average molecular weight; LMT of 1.9–12% < 50 kDa (mean 7%)), almost complying with the EU specification, the no‐observed‐adverse‐effect level (NOAEL) was equal to 3,394–3,867 mg/kg bw per day for males and females, respectively, the highest dose tested;
- no adverse effects have been detected in several chronic toxicity studies in rats with carrageenan (mostly κ/λ‐type, no adequate indication of molecular weight distribution); from the available rat studies, NOAELs up to 7,500 mg/kg bw per day, the highest dose tested, were identified (Nilson and Wagner, 1959). However, in another study in rats given carrageenan preparations with a molecular weight of 244 and 252 kDa, a lowest‐observed‐adverse‐effect level (LOAEL) of 1% in the diet (equivalent to 500 mg/kg bw per day) was identified by the Panel (Documentation provided to EFSA n. 43). The Panel noted that the characterisation of the test material in all the chronic studies was limited;
- carrageenan (different types) and processed Eucheuma seaweed did not raise a concern with respect to genotoxicity;
- there was no concern with respect to carcinogenicity for carrageenan (mostly κ/λ‐type);
- the NOAEL of sodium and calcium κ/λ‐carrageenan for developmental effects found in dietary prenatal developmental toxicity studies was 3,000 and 5,000 mg/kg bw per day for rats and hamsters (Documentation provided to EFSA n. 46), and 3,060 mg calcium carrageenan/kg bw per day for rats (Collins et al., 1977a), the highest doses tested;
- the Panel considered that for the safety evaluation of processed Eucheuma seaweed (E 407a) read across can be made from study results used in the toxicological evaluation of carrageenan (E 407).
In addition, the Panel observed that:
- from all the data received, data were adequate for a refined exposure assessment for 41 out of 79 food categories;
- in the general population, based on the reported use levels, a refined exposure, in the brand‐loyal scenario of up to 758.6 mg/kg bw per day for toddlers (from 12 months up to and including 35 months of age) was estimated;
- for populations consuming foods for special medical purposes and special formulae, the 95th percentile of maximum exposure assessments calculated based on the maximum reported data from food industry (equal to the MPL) were up to 49.4 mg/kg bw per day for infants (from 12 weeks up to and including 11 months of age);
For the food additives E 407 and E 407a, the fraction of low molecular weight carrageenan is limited in the EC specifications by the purity criteria. The Panel was informed by one interested party that the material on the market does not necessarily comply with the EU specifications regarding the limit of the low molecular weight fraction. This fraction has been associated with potential health hazards similar to those reported for preparations of degraded carrageenan, such as poligeenan or C16, to which this fraction shows similarity in molecular structure and in weight‐average molecular weight. Although, full identity of degraded carrageenan such as poligeenan or C16 with the low molecular weight fraction of carrageenan has not been specifically demonstrated.
Concerning degraded carrageenan (e.g. poligeenan, C16), the Panel therefore noted the following:
- degraded carrageenan has been described to be absorbed and to be present in various tissues, namely the liver, and the urine of animals when administered in drinking water or via the diet;
- degraded carrageenan did not raise concern with respect to genotoxicity;
- rats exposed to degraded ι‐carrageenan (weight‐average molecular weight of 20–40 kDa) via drinking water, diet or by gavage for up to 24 months developed in first instance colitis, secondary metaplasia and finally tumours (squamous cell carcinomas, adenocarcinomas, adenomas);
- monkeys given degraded ι‐carrageenan (C16) via drinking water showed histopathological lesions in the colon which varied from slight mucosal erosions at the low dose (750 mg/kg bw per day) to ulceration associated with inflammatory infiltration of the lamina propria at the high dose (2,900 mg/kg bw per day). In this study, all monkeys on C16 lost blood frequently from the intestinal tract in a dose‐related degree and developed some degree of anaemia. A LOAEL of 750 mg degraded ι‐carrageenan (C16)/kg bw per day was noted;
- toxicity studies have been mainly conducted with degraded ι‐carrageenan, thus, degraded κ‐ and λ‐carrageenan being sparsely toxicologically characterised.
The following uncertainties were noted by the Panel as regards the chemistry and fate of carrageenan (E 407) and processed Eucheuma seaweed (E 407a):
- no data are available showing the molecular weight distribution for different food‐grade carrageenan preparations within the defined weight‐average molecular weight range of 200–800 kDa;
- no data are available showing the molecular weight distribution for individual food‐grade processed Eucheuma seaweed preparations;
- the weight‐average molecular weight range of carrageenan (E 407) and processed Eucheuma seaweed (E 407a) is not defined in the EU specifications allowing for the presence of a low weight‐average molecular weight fraction of carrageenan. Based on the information provided on weight‐average molecular weights of commercially available carrageenans, the low molecular weight material needs to be accurately quantified;
- in most of the toxicological studies the carrageenan used is not well specified and its weight‐average molecular weight and its content of low molecular weight fragments are not given;
- although it has been claimed that there is no adequate analytical method available to measure the low molecular weight fraction, the Panel noted that there is indication of the percentage of the low molecular weight fraction in the food additive carrageenan (E 407) tested in a few toxicological studies;
- only limited information on the stability of carrageenan in food was available. No data on stability of carrageenan and/or processed Eucheuma seaweed addressing the usual variation of parameters (temperature, pH) relevant for the authorised food uses were available. Information on possible degradation products under acidic conditions in relevant food products is missing;
- studies investigating the hydrolysis of the κ‐, ι‐ and λ‐carrageenan showed contradictory results.
The Panel further noted the following uncertainty as regards the exposure assessment scenario:
- the refined estimates were based on 41 out of 79 food categories in which carrageenan (E 407) and processed Eucheuma seaweed (E 407a) are authorised and result in an overestimation of the real exposure to carrageenan (E 407) and processed Eucheuma seaweed (E 407a) as food additives in European countries considering only food additives uses for which data have been provided were considered.
Among the uncertainties from the biological and toxicological data, the Panel considered the following:
- the lack of reliable comparative toxicokinetic and toxicological studies between the different types of carrageenan and their corresponding low molecular weight fractions;
- the theoretical possibility that limited degradation could occur under conditions representative of the in vivo situation;
- no firm conclusion on the other types of carrageenan could be drawn on the observation of occult blood in faeces of rhesus monkeys dosed with a commercial carrageenan;
- the characterisation of the test material in most of the toxicological studies was limited;
- there were no adequate toxicological studies performed with low weight‐average molecular weight carrageenan (around 200 kDa), apart from one 90‐day study (with an average molecular weight carrageenan in the range of 196–257 kDa, not specified if it was a number‐average or a weight‐average);
- testing for chronic toxicity and reproductive and developmental toxicity was performed almost exclusively with κ/λ‐carrageenan; almost no data on ι‐carrageenan were available;
- inadequate data on the possible relevance of carrageenan exposure for existing inflammatory bowel diseases in humans;
- the relevance for humans of the observations in animal studies pointing to the induction of glucose intolerance and glucosuria by carrageenan is unclear;
- the possible role of sulfate and the interactions of the various forms of carrageenans with the gut microflora in some of the reported inflammatory effects of carrageenans.
Altogether in most toxicological studies, the molecular weight distribution of the carrageenan tested is not or only inadequately described. The low molecular weight fraction of carrageenan is associated with potential adverse effects. This is due to its similarity in molecular structure and weight‐average molecular weight with those of degraded carrageenan, such as poligeenan or C16, known to cause inflammatory intestinal effects. Moreover, results suggesting that carrageenan might enhance inflammatory bowel disease in humans need clarification. The test compounds used in a large number of the toxicity tests are thus not considered to reflect adequately the diversity of preparations of the food additive on the market, particularly with respect to the broad range of weight‐average molecular weights reported. The Panel noted that most of these issues were also raised and discussed in a recent review (David et al., 2018).
The Panel also considered other pending uncertainties regarding the relevance of the studies available to assess the safety of the authorised food additive carrageenan (E 407). The physicochemical properties of carrageenan depend on the chemical conformation (helical or random coil) in which it exists in the preparations and in foods (solid or liquids foods) and are influenced by the presence of cations, proteins and the pH and temperature. These conditions could affect the toxicity of carrageenan and are thus relevant for the safety assessment of the food additive in the authorised uses. Furthermore, findings in some studies suggest that carrageenan exposure could be related to the induction of glucose intolerance and glucosuria. In the view of the Panel, all this information still needs clarification.
Overall, taking into account the lack of adequate data to address the above‐mentioned uncertainties, the Panel concluded that the existing group ADI for carrageenan (E 407) and processed Eucheuma seaweed (E 407a) of 75 mg/kg bw per day should be considered temporary, while the database should be improved within 5 years after publication of this opinion. Within the given time frame, high importance should be ascribed to the establishment of an adequate interlaboratory validated analytical method to quantify, at the existing 5% limit, the low weight‐average molecular weight fraction of carrageenan, and to establish whether or not this fraction is associated with health risks.
The Panel further concluded that in the refined brand‐loyal exposure assessment scenario the exposure estimates exceeded, in some cases by up to approximately 10‐fold, the temporary existing ADI at the high level (95th percentile) for all population groups and at the mean for all population groups except for infants and the elderly. Although the exposure may be overestimated, the extent of the exceedance of the ADI (10‐fold) may be a safety concern.
The Panel recommended that the EC considers:
- in the definition for the food additives E 407 and E 407a in the Commission Regulation (EU) No 231/2012, specifying the weight‐average molecular weight range in a narrow way avoiding a significant overlap with the molecular weight range of preparations of degraded carrageenan;
- with respect to the purity criteria in the Commission Regulation (EU) No 231/2012, the need of an interlaboratory validated analytical method to detect low molecular weight carrageenan in the food additives E 407 and E 407a at the limit specified in the Regulation;
- possibility of extending the molecular weight of the existing 5% limit for low molecular weight carrageenan from 50 kDa to up to 200 kDa for the food additives E 407 and E 407a in the Commission Regulation (EU) No 231/2012. This would take into account the limitation of the available analytical methods to quantify the percentage of low weight‐average molecular weight carrageenan and will further make certain that the presence of low weight‐average molecular weight carrageenan is at a low level; it should be indicated if the 5% limit refers to the food additive as such or on dried basis.
- the need of information on the stability of the food additives E 407 and E 407a in food. No data on stability of carrageenan (E 407) and/or processed Eucheuma seaweed (E 407a) addressing the usual variation of parameters (temperature, pH) relevant for the authorised food uses were available. Information on possible degradation products under acidic conditions in relevant food products was missing;
- revising the maximum limits for the impurities of toxic elements (lead, mercury, cadmium and arsenic) in the EC specification for carrageenan (E 407) and processed Eucheuma seaweed (E 407a) in order to ensure that the food additives will not be a significant source of exposure to these toxic elements in food;
- collecting monitoring data for the food categories contributing the most to the exposure to carrageenan (E 407) and processed Eucheuma seaweed (E 407a) in order to allow for more refined estimates of exposure;
- establishing, as a general principle, numerical MPLs for all food authorised uses with special consideration of the main food categories contributing to the exposure (e.g. flavoured milk products, flavoured drinks, food supplements and fine bakery wares) to carrageenan (E 407) and processed Eucheuma seaweed (E 407a) as food additives in order to reduce consumers exposure.