Oxidative Stress, Mutations and Chromosomal Aberrations Induced by In Vitro and In Vivo Exposure to Furan.

Furan is a volatile compound that is formed in foods during thermal processing. It is classified as a possible human carcinogen by international authorities based on sufficient evidence of carcinogenicity from studies in experimental animals. Although a vast number of studies both in vitro and in vivo have been performed to investigate furan genotoxicity, the results are inconsistent, and its carcinogenic mode of action remains to be clarified. Here, we address the mutagenic and clastogenic activity of furan and its prime reactive metabolite cis-2 butene-1,4-dial (BDA) in mammalian cells in culture and in mouse animal models in a search for DNA lesions responsible of these effects. To this aim, Fanconi anemia-derived human cell lines defective in the repair of DNA inter-strand crosslinks (ICLs) and Ogg1-/- mice defective in the removal of 8-hydroxyguanine from DNA, were used. We show that both furan and BDA present a weak (if any) mutagenic activity but are clear inducers of clastogenic damage. ICLs are strongly indicated as key lesions for chromosomal damage whereas oxidized base lesions are unlikely to play a critical role.

Free radical scavenging capacity and protective effect of natural substances in peloids from the thermal spring pool Bagnaccio (Viterbo, Italy).

Natural peloids from sulfurous thermal springs are largely used in cosmetic and pelotherapy for the treatment of different dermatological conditions, including skin aging, dermatitis, and other eczemas. The beneficial effects are correlated to mineralogical and other thermal properties, as well as to the presence of natural substances with specific antioxidant activity. Few data are available for the comparison between natural peloids and synthetic (i.e., artificially maturated) muds. In this context, the natural substances and antioxidant activity of natural white mud (WM) and dark mud (DM) peloids from the sulfurous thermal spring pool Bagnaccio (Viterbo, Italy) have been studied in detail to evaluate possible relationships between physicochemical properties and therapeutic effect. A large panel of natural substances in WM and DM were characterized for the first time by ³¹P-nuclear magnetic resonance and gas chromatography associated to mass spectrometry analysis. Polar fractions of WM and DM peloids were characterized by the presence of several bioactive natural compounds, showing high antioxidant activity and DNA protective effect, as evaluated by 2,2-diphenyl-1-picrylhydrazyl assay, and hydrogen peroxide­induced DNA breakage in the alkaline comet assay. The antioxidant activity and DNA protective effect could be attributed to radical scavenging rather than a modulatory effect on the induced DNA repair, and are of order of intensity higher than that reported for synthetic muds.

Ultrasound driven assembly of lignin into microcapsules for storage and delivery of hydrophobic molecules.

Oil-filled microcapsules of kraft lignin were synthesized by first creating an oil in water emulsion followed by a high-intensity, ultrasound-assisted cross-linking of lignin at the water/oil interface. The rationale behind our approach is based on promoting documented lignin hydrophobic interactions within the oil phase, followed by locking the resulting spherical microsystems by covalent cross-linking using a high intensity ultrasound treatment. As further evidence in support of our rationale, confocal and optical microscopies demonstrated the uniformly spherical morphology of the created lignin microparticles. The detailed elucidation of the cross-linking processes was carried out using gel permeation chromatography (GPC) and quantitative (31)P NMR analyses. The ability of lignin microcapsules to incorporate and release Coumarin-6 was evaluated in detail. In vitro studies and confocal laser scanning microscopy analysis were carried out to assess the internalization of capsules into Chinese hamster ovary (CHO) cells. This part of our work demonstrated that the lignin microcapsules are not cytotoxic and readily incorporated in the CHO cells.

Effect of blueberries (BB) on micronuclei induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 7,12-dimethylbenz(a)anthracene (DMBA) in mammalian cells, assessed in in vitro and in vivo assays.

The protective effect of blueberry (BB) on the clastogenic effects of MNNG and DMBA was evaluated with the induced micronucleus (MN) frequency as a biomarker, both in vitro and in vivo. Human hepatoma HepG2 cells, which contain most of the metabolic activating enzymes was used for the in vitro test. MN frequencies were determined in binucleated cells generated by blocking cytokinesis by use of cytochalasin-B. The MN frequency in vivo was determined in polychromatic erythrocytes (PCEs) from the bone marrow of treated mice. BB by itself was not toxic both in vivo and in vitro. There was no evidence of a potential physico-chemical interaction between BB and the test carcinogens in vitro. Pre-treatment with BB reduced the MN frequency induced by MNNG. But, simultaneous treatment and post-treatment with BB did not affect the frequency of MNNG-induced MN. BB did not affect the frequency of DMBA-induced MN in vitro under any test condition. Under in vivo conditions, BB reduced the frequencies of MNNG- and DMBA-induced MN in PCEs, but in the case of the protective effect of BB against DMBA a dramatic reduction in the percentage of PCEs was observed, suggesting increased cytotoxicity.

The use of cyprinodont fish, Aphanius fasciatus, as a sentinel organism to detect complex genotoxic mixtures in the coastal lagoon ecosystem.

In the present work we aimed to standardise the alkaline comet assay with erythrocytes of the cyprinodont, Mediterranean Killifish, Aphanius fasciatus. The aims of the study were to explore the suitability of this fish to assess biomarkers of genotoxic effects and as a sentinel organism to detect complex genotoxic mixtures in coastal lagoon ecosystems. Following proper optimisation, the application and effectiveness of the comet assay in erythrocytes of A. fasciatus were tested by measuring the tail DNA (%) induced by (a) in vivo exposure of individual fish to X-rays (dose, 3Gy) and (b) following in vitro challenge of erythrocytes with restriction endonucleases Fok-I and Eco-RI, which selectively induce double-strand breaks with cohesive and blunt termini, respectively. Furthermore, in order to evaluate whether circulating fish blood contained actively proliferating cells that could influence the extent of DNA damage in control (untreated) fish, we measured the number of "comets" positive for 5-bromo-2'-deoxyuridine (BrdU) by the use of anti-BrdU antibody and immuno-histochemical methods. Both treatments (i.e. with X-rays and restriction endonucleases) induced statistically significant increases in tail DNA (%) values compared with the relevant untreated controls, indicating the effectiveness of the comet assay in the erythrocytes of A. fasciatus to detect different types of DNA lesions. Results from anti-BrdU antibody labelling of erythrocytes indicated a very low percentage (5%) of "comets" positive for BrdU. Following optimisation and validation of the assay under laboratory conditions, fish were collected in the Orbetello lagoon (Tuscany, Italy), considered to be a significantly polluted site. The results showed statistically significant increases for tail DNA (%) compared with corresponding values observed in erythrocytes of fish caught in the unpolluted reference site "Saline di Tarquinia". The effects of physico-chemical parameters of the water (i.e., salinity, pH and oxygen content) did not significantly influence the induction of DNA damage. These results indicate that the comet assay provides a reliable parameter and that A. fasciatus is a promising "sentinel organism" to detect the genotoxic impact of complex mixtures in coastal lagoon ecosystems.

Relationship between chromatin structure, DNA damage and repair following X-irradiation of human lymphocytes.

Earlier studies using the technique of premature chromosome condensation (PCC) have shown that in human lymphocytes, exchange type of aberrations are formed immediately following low doses (<2 Gy) of X-rays, whereas at higher doses these aberrations increase with the duration of recovery. This reflects the relative roles of slow and fast repair in the formation of exchange aberrations. The underlying basis for slow and fast repairing components of the DNA repair may be related to differential localization of the initial damage in the genome, i.e., between relaxed and condensed chromatin. We have tried to gain some insight into this problem by (a) X-irradiating lymphocytes in the presence of dimethyl sulfoxide (DMSO) a potent scavenger of radiation-induced .OH radicals followed by PCC and (b) probing the damage and repair in two specific chromosomes, 18 and 19, which are relatively poor and rich in transcribing genes by COMET-FISH, a combination of Comet assay and fluorescence in situ hybridization (FISH) techniques. Results obtained show (a) that both fast appearing and slowly formed exchange aberrations seem to take place in relaxed chromatin, since they are affected to a similar extent by DMSO, (b) significant differential DNA breakage of chromosome 18 compared to chromosome 19 in both G0 and G1 phases of the cell cycle as detected by Comet assay, indicating that relaxed chromatin containing high densities of transcriptionally active genes shows less fragmentation due to fast repair (chromosome 19) compared to chromosome 18, and (c) that relaxed chromatin is repaired or mis-repaired faster than more compact chromatin.

A selective de-O-methylation of guaiacyl lignans to corresponding catechol derivatives by 2-iodoxybenzoic acid (IBX). The role of the catechol moiety on the toxicity of lignans.

We report here the first selective de-O-methylation of a large panel of guaiacyl lignans to the corresponding catechol derivatives by using IBX as primary oxidant under green conditions (dimethyl carbonate-H(2)O solvent) through an in situ reduction procedure. The influence of the catechol moiety on the cytotoxicity and genotoxicity of new lignan derivatives has been investigated. The results obtained indicated that the presence of the catechol moiety sharply enhances the clastogenic potential (e.g. induction of chromosomal aberrations), the cytotoxicity and the modulation of cell cycle progression with respect to the parent compounds. Thus, despite the in vitro antioxidant activity usually described for catechol derivatives, our results show for the first time the generation of a clastogenic potential, highly indicative of a long-term genetic and cancer risk.

In Vitro Cytogenetic Assays: Chromosomal Aberrations and Micronucleus Tests.

Chromosome damage is a very important indicator of genetic damage relevant to environmental and clinical studies. Detailed descriptions of the protocols used for detection of chromosomal aberrations induced by genotoxic agents in vitro both in the presence or absence of rat liver-derived metabolizing systems are given in this chapter. Structural chromosomal aberrations that can be observed and quantified at metaphases are described here. For the detection of chromosomal damage (fragments or whole chromosome) in interphase, the micronucleus test can be used, and a description of this test is also presented. Criteria for determining a positive result using appropriate statistical methods are described.

Genotoxicity assessment of chemical mixtures.

The EFSA Scientific Committee addressed in this document the peculiarities related to the genotoxicity assessment of chemical mixtures. The EFSA Scientific Committee suggests that first a mixture should be chemically characterised as far as possible. Although the characterisation of mixtures is relevant also for other toxicity aspects, it is particularly significant for the assessment of genotoxicity. If a mixture contains one or more chemical substances that are individually assessed to be genotoxic in vivo via a relevant route of administration, the mixture raises concern for genotoxicity. If a fully chemically defined mixture does not contain genotoxic chemical substances, the mixture is of no concern with respect to genotoxicity. If a mixture contains a fraction of chemical substances that have not been chemically identified, experimental testing of the unidentified fraction should be considered as the first option or, if this is not feasible, testing of the whole mixture should be undertaken. If testing of these fraction(s) or of the whole mixture in an adequately performed set of in vitro assays provides clearly negative results, the mixture does not raise concern for genotoxicity. If in vitro testing provides one or more positive results, an in vivo follow-up study should be considered. For negative results in the in vivo follow-up test(s), the possible limitations of in vivo testing should be weighed in an uncertainty analysis before reaching a conclusion of no concern with respect to genotoxicity. For positive results in the in vivo follow-up test(s), it can be concluded that the mixture does raise a concern about genotoxicity.

In vitro cytogenetic results supporting a DNA nonreactive mechanism for ochratoxin A, potentially relevant for its carcinogenicity.

Ochratoxin A (OTA) is a widespread mycotoxin of cereals and many agricultural products and causes high incidences of renal tumors in rodents. Although its carcinogenic properties have been known since the eighties, the precise mechanism of action is still relatively undefined. At present, increasing evidence suggests that OTA does not act with a direct genotoxic mechanism, opposed to other previous evidence where the formation of DNA adducts by 32P-postlabeling was observed. The genotoxic activity of OTA assessed in a variety of in vitro and in vivo studies was very low if genotoxic at all. In this study, we clearly show that OTA does not bear any clastogenic or aneugenic activity based on the absence of the induction of chromosome aberrations, sister chromatid exchanges, and micronuclei in human lymphocytes and V79 cells in vitro in both the absence and the presence of S9 metabolism. Alternatively, cytogenetic analyses evidenced significant increases in endoreduplicated cells and highly condensed metaphases with separated chromatids. This implies that OTA or its possible metabolites do not covalently bind DNA through the formation of adducts since structural chromosome aberrations are a very sensitive end points to detect chemical carcinogens with electrophilic substituents. Alternatively, induction of endoreduplication and chromatid separation provides strong evidence for a DNA nonreactive mechanism of OTA carcinogenicity involving the disruption of mitosis by interfering with key regulators of chromosome separation and progression of mitosis. This causes a temporary arrest of mitoses and premature exit from it (mitotic slippage) to generate endoreduplication and polyploidy accompanied by increased risk of aneuploidy and subsequent tumor formation.

Re-evaluation of silicon dioxide (E 551) as a food additive.

The EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion re-evaluating the safety of silicon dioxide (E 551) when used as a food additive. The forms of synthetic amorphous silica (SAS) used as E 551 include fumed silica and hydrated silica (precipitated silica, silica gel and hydrous silica). The Scientific Committee on Food (SCF) established a group acceptable daily intake (ADI) 'not specified' for silicon dioxide and silicates. SAS materials used in the available biological and toxicological studies were different in their physicochemical properties; their characteristics were not always described in sufficient detail. Silicon dioxide appears to be poorly absorbed. However, silicon-containing material (in some cases presumed to be silicon dioxide) was found in some tissues. Despite the limitations in the subchronic, reproductive and developmental toxicological studies, including studies with nano silicon dioxide, there was no indication of adverse effects. E 551 does not raise a concern with respect to genotoxicity. In the absence of a long-term study with nano silicon dioxide, the Panel could not extrapolate the results from the available chronic study with a material, which does not cover the full-size range of the nanoparticles that could be present in the food additive E 551, to a material complying with the current specifications for E 551. These specifications do not exclude the presence of nanoparticles. The highest exposure estimates were at least one order of magnitude lower than the no observed adverse effect levels (NOAELs) identified (the highest doses tested). The Panel concluded that the EU specifications are insufficient to adequately characterise the food additive E 551. Clear characterisation of particle size distribution is required. Based on the available database, there was no indication for toxicity of E 551 at the reported uses and use levels. Because of the limitations in the available database, the Panel was unable to confirm the current ADI 'not specified'. The Panel recommended some modifications of the EU specifications for E 551.

Safety of hydroxyanthracene derivatives for use in food.

The Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion on the safety of hydroxyanthracene derivatives and to provide advice on a daily intake that does not give rise to concerns about harmful effects to health. Hydroxyanthracene derivatives are a class of chemical substances naturally occurring in different botanical species and used in food to improve bowel function. The ANS Panel reviewed the available scientific data on a possible relationship between hydroxyanthracene derivatives exposure and genotoxic and carcinogenic effects. On the basis of the data currently available, the Panel noted that emodin, aloe-emodin and the structurally related substance danthron have shown evidence of in vitro genotoxicity. Aloe extracts have also been shown to be genotoxic in vitro possibly due to the presence of hydroxyanthracene derivatives in the extract. Furthermore, aloe-emodin was shown to be genotoxic in vivo and the whole-leaf aloe extract and the structural analogue danthron were shown to be carcinogenic. Epidemiological data suggested an increased risk for colorectal cancer associated with the general use of laxatives, several of which contain hydroxyanthracene derivatives. Considering the possible presence of aloe-emodin and emodin in extracts, the Panel concluded that hydroxyanthracene derivatives should be considered as genotoxic and carcinogenic unless there are specific data to the contrary, such as for rhein, and that there is a safety concern for extracts containing hydroxyanthracene derivatives although uncertainty persists. The Panel was unable to provide advice on a daily intake of hydroxyanthracene derivatives that does not give rise to concerns about harmful effects to health.

Scientific Opinion of Flavouring Group Evaluation 411 (FGE.411): 2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-(thiophen-2-ylmethyl)acetamide from chemical group 30 (miscellaneous substances).

EFSA was requested to deliver a scientific opinion on the implications for human health of the flavouring substance 2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-(thiophen-2-ylmethyl)acetamide [FL-no: 16.133], in the Flavouring Group Evaluation 411 (FGE.411), according to Regulation (EC) No 1331/2008 of the European Parliament and of the Council. The substance has not been reported to occur in natural source materials of botanical or animal origin. It is intended to be used as a flavouring substance in specific categories of food but not intended to be used in beverages, except for milk and dairy based beverages that are opaque. The chronic dietary exposure to the substance estimated using the added portions exposure technique (APET), is calculated to be 225 µg/person per day for a 60-kg adult and 142 µg/person per day for a 15-kg 3-year-old child. A 90-day oral gavage study in rats showed no adverse effects at doses up to 100 mg/kg body weight (bw) per day, providing an adequate margin of safety. Developmental toxicity was not observed in a study with rats at the dose levels up to 1,000 mg/kg bw per day. The Panel concluded that there is no safety concern for [FL-no: 16.133], when used as a flavouring substance at the estimated level of dietary exposure calculated using the APET approach and based on the recommended uses and use levels as specified in Appendix  B. This conclusion does not apply for use in beverages where the substance can be subject to phototransformation.

Scientific Opinion on Flavouring Group Evaluation 200, Revision 1 (FGE.200 Rev.1): 74 α,ß-unsaturated aliphatic aldehydes and precursors from chemical subgroup 1.1.1 of FGE.19.

The Panel on Food Additives and Flavourings of the European Food Safety Authority was requested to evaluate the genotoxic potential of 74 flavouring substances from subgroup 1.1.1 of FGE.19 in the Flavouring Group Evaluation 200 Revision 1 (FGE.200 Rev1). In FGE.200, genotoxicity studies were provided for one representative substance, namely hex-2(trans)-enal [FL-no: 05.073], and for other two substances in the same subgroup, namely 2-dodecenal [FL-no: 05.037] and 2-nonenal [FL-no: 05.171]. The Panel concluded that the concern still remains with respect to genotoxicity for the substances of this subgroup and requested an in vivo Comet assay performed in duodenum and liver for hex-2(trans)-enal [FL-no: 05.073]. For the two other representative substances of subgroup 1.1.1 (nona-2(trans),6(cis)-dienal [FL-no: 05.058] and oct-2-enal [FL-no: 05.060]), the Panel requested a combined in vivo Comet assay and micronucleus assay. These data have been provided and are evaluated in the present opinion FGE.200 Rev1. Industry submitted genotoxicity studies on trans-2-octenal [FL-no: 05.190], instead of oct-2-enal [FL-no: 05.060]. Based on the available data, the Panel concluded that the concern for genotoxicity can be ruled out for hex-2(trans)-enal [FL-no: 05.073], trans-2-octenal [FL-no: 05.190] and nona-2(trans),6(cis)-dienal [FL-no: 05.058], therefore all the 74 substances [FL-no: 02.020, 02.049, 02.050, 02.090, 02.112, 02.137, 02.156, 02.192, 02.210, 02.231, 05.037, 05.058, 05.060, 05.070, 05.072, 05.073, 05.076, 05.078, 05.102, 05.109, 05.111, 05.114, 05.120, 05.144, 05.150, 05.171, 05.172, 05.179, 05.184, 05.189, 05.190, 05.191, 05.195, 06.025, 06.031, 06.072, 09.054, 09.097, 09.109, 09.119, 09.146, 09.233, 09.244, 09.247, 09.276, 09.277, 09.303, 09.312, 09.385, 09.394, 09.395, 09.396, 09.397, 09.398, 09.399, 09.400, 09.410, 09.411, 09.469, 09.482, 09.489, 09.492, 09.493, 09.498, 09.678, 09.701, 09.719, 09.741, 09.790, 09.841, 09.866, 09.947, 09.948, 13.004] can be evaluated through the Procedure for flavouring substances.

Scientific Opinion on Flavouring Group Evaluation 201 Revision 2 (FGE.201Rev2): 2-alkylated, aliphatic, acyclic alpha,beta-unsaturated aldehydes and precursors, with or without additional double-bonds, from chemical subgroup 1.1.2 of FGE.19.

The Panel on Food Additives and Flavourings of the European Food Safety Authority was requested to consider in this revision 2 of Flavouring Group Evaluation 201, the additional data on genotoxicity submitted by the Industry on two substances, 2-methylpent-2-enal [FL-no: 05.090] and 2 methylcrotonaldehyde [FL-no: 05.095], from subgroup 1.1.2 of FGE.19. In FGE.201Rev1, the Panel concluded that further data were required in order to clarify the genotoxic potential of this subgroup and considered the testing of 2-methylcrotonaldehyde [FL-no: 05.095] in a comet assay in liver and duodenum, the first site of contact, as a preferred option to further investigate the genotoxicity in vivo. New genotoxicity studies have been submitted for both 2-methylpent-2-enal [FL-no: 05.090] and 2-methylcrotonaldehyde [FL-no: 05.095]. 2-Methylpent-2-enal [FL-no: 05.090] tested in a combined micronucleus/comet assay did not induce DNA damage, overruling the weak gene mutation effect observed in bacteria and confirming the negative results observed in the in vitro micronucleus assay. 2-Methylcrotonaldehyde [FL-no: 05.095] did not induce gene mutations in liver and glandular stomach of transgenic rats. In addition, 2-methylcrotonaldehyde [FL-no: 05.095] tested in an in vivo comet assay in liver and duodenum, it did not induce DNA damage. Overall, the Panel concluded that the genotoxic evidence observed in vitro, was not confirmed in vivo for the representative substances 2-methylcrotonaldehyde [FL-no: 05.095] and 2-methylpent-2-enal [FL-no: 05.090], therefore all the 10 substances in this subgroup [FL-no: 02.174, 05.033, 05.090, 05.095, 05.105, 05.107, 05.126, 07.261, 12.065 and 12.079] can be evaluated through the Procedure for the evaluation of flavouring substances.

Scientific Opinion of Flavouring Group Evaluation 406 (FGE.406): (S)-1-(3-(((4-amino-2,2-dioxido-1H-benzo[c][1,2,6]thiadiazin-5-yl)oxy)methyl)piperidin-1-yl)-3-methylbutan-1-one.

The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) of EFSA was requested to deliver a scientific opinion on the safety of the use of the substance (S)-1-(3-(((4-amino-2,2-dioxido-1H-benzo[c][1,2,6]thiadiazin-5-yl)oxy)methyl)piperidin-1-yl)-3-methylbutan-1-one [FL-no: 16.129], as a flavouring substance. The substance is intended to be used in the form of its sodium salt as a flavour modifier in beverages. The Panel concluded that [FL-no: 16.129] would not raise a concern with respect to genotoxicity under conditions where it remains stable and does not undergo photodegradation. However, the data provided do not rule out genotoxicity for the degradation products. A 90-day toxicity study with [FL-no: 16.129] in rats showed no adverse effects at exposure up to 100 mg/kg body weight (bw) per day. No developmental toxicity was observed in rats at dose levels up to 1,000 mg/kg bw per day. An adequate margin of safety was calculated for [FL-no: 16.129]. The Panel concluded that [FL-no: 16.129] and its sodium salt are not expected to be of safety concern at the estimated levels of intake. This conclusion applies only to the use of the substance as a flavour modifier at levels up to those specified in beverages, but not to the degradation products that may be formed upon exposure to ultraviolet-A (UV-A) light. The conditions protecting [FL-no: 16.129] from photodegradation have not been adequately investigated. It is also unclear if degradation occurs in the absence of UV light. Based on the data provided, the Panel cannot conclude on the safety of [FL-no: 16.129] when used as a flavour modifier.

Re-evaluation of sodium, potassium and calcium salts of fatty acids (E 470a) and magnesium salts of fatty acids (E 470b) as food additives.

The EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion re-evaluating the safety of sodium, potassium and calcium salts of fatty acids (E 470a) and magnesium salts of fatty acids (E 470b) when used as food additives. In 1991, the Scientific Committee on Food (SCF) established a group acceptable daily intake (ADI) 'not specified' for the fatty acids (myristic-, stearic-, palmitic- and oleic acid) and their salts. The sodium, potassium, calcium and magnesium salts of fatty acids are expected to dissociate in the gastrointestinal tract to fatty acid carboxylates and their corresponding cations. There were no data on subchronic toxicity, chronic toxicity, reproductive and developmental toxicity of the salts of fatty acids. There was no concern for mutagenicity of calcium caprylate, potassium oleate and magnesium stearate. From a carcinogenicity study with sodium oleate, a no observed adverse effect level (NOAEL) could not be identified but the substance was considered not to present a carcinogenic potential. Palmitic- and stearic acid which are the main fatty acids in E 470a and E 470b were already considered of no safety concern in the re-evaluation of the food additive E 570. The fatty acid moieties of E 470a and E 470b contributed maximally for 5% to the overall intake of saturated fatty acids from all dietary sources. Overall, the Panel concluded that there was no need for a numerical ADI and that the food additives sodium, potassium, calcium and magnesium salts of fatty acids (E 470a and E 470b) were of no safety concern at the reported uses and use levels.

Scientific opinion on flavouring group evaluation 77, revision 3 (FGE.77Rev3): consideration of pyridine, pyrrole and quinoline derivatives evaluated by JECFA (63rd meeting) structurally related to pyridine, pyrrole, indole and quinoline derivatives evaluated by EFSA in FGE.24Rev2.

The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids of the EFSA was requested to consider evaluations of flavouring substances assessed since 2000 by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and to decide whether further evaluation is necessary, as laid down in Commission Regulation (EC) No 1565/2000. The present consideration concerns a group of 22 pyridine, pyrrole and quinoline derivatives evaluated by JECFA (63rd meeting). The revision of this consideration is made since additional genotoxicity data have become available for 6-methylquinoline [FL-no: 14.042]. The genotoxicity data available rule out the concern with respect to genotoxicity and accordingly the substance is evaluated through the Procedure. For all 22 substances [FL-no: 13.134, 14.001, 14.004, 14.007, 14.030, 14.038, 14.039, 14.041, 14.042, 14.045, 14.046, 14.047, 14.058, 14.059, 14.060, 14.061, 14.065, 14.066, 14.068, 14.071, 14.072 and 14.164] considered in this Flavouring Group Evaluation (FGE), the Panel agrees with the JECFA conclusion, 'No safety concern at estimated levels of intake as flavouring substances' based on the Maximised Survey-derived Daily Intake (MSDI) approach. Besides the safety assessment of these flavouring substances, the specifications for the materials of commerce have also been evaluated, and the information is considered adequate for all the substances. For the following substances [FL-no: 13.134, 14.001, 14.030, 14.041, 14.042, 14.058, 14.072], the Industry has submitted use levels for normal and maximum use. For the remaining 15 substances, use levels are needed to calculate the modified Theoretical Added Maximum Daily Intakes (mTAMDIs) in order to identify those flavouring substances that need more refined exposure assessment and to finalise the evaluation.

Re-evaluation of propane-1,2-diol (E 1520) as a food additive.

The EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion re-evaluating the safety of propane-1,2-diol (E 1520) when used as a food additive. In 1996, the Scientific Committee on Food (SCF) established an acceptable daily intake (ADI) of 25 mg/kg body weight (bw) per day for propane-1,2-diol. Propane-1,2-diol is readily absorbed from the gastrointestinal and is expected to be widely distributed to organs and tissues. The major route of metabolism is oxidation to lactic acid and pyruvic acid. At high concentrations, free propane-1,2-diol is excreted in the urine. No treatment-related effects were observed in subchronic toxicity studies. The available data did not raise concern with respect to genotoxicity. Haematological changes suggestive of an increased red blood cell destruction with a compensatory increased rate of haematopoiesis were observed at the highest dose level (5,000 mg/kg bw per day) in a 2-year study in dogs. No adverse effects were reported in a 2-year chronic study in rats with propane-1,2-diol (up to 2,500 mg/kg bw per day). The SCF used this study to derive the ADI. No adverse effects were observed in the available reproductive and developmental toxicity studies. Propane-1,2-diol (E 1520) is authorised according to Annex III in some food additives, food flavourings, enzymes and nutrients and it is then carried over to the final food. Dietary exposure to E 1520 was assessed based on the use levels and analytical data. The Panel considered that for the food categories for which information was available, the exposure was likely to be overestimated. Considering the toxicity database, the Panel concluded that there was no reason to revise the current ADI of 25 mg/kg bw per day. The Panel also concluded that the mean and the high exposure levels (P95) of the brand-loyal refined exposure scenario did not exceed the ADI in any of the population groups from the use of propane-1,2-diol (E 1520) at the reported use levels and analytical results.

Re-evaluation of carrageenan (E 407) and processed Eucheuma seaweed (E 407a) as food additives.

The present opinion deals with the re-evaluation of the safety of food-grade carrageenan (E 407) and processes Eucheuma seaweed (E 407a) used as food additives. Because of the structural similarities, the Panel concluded that processed Eucheuma seaweed can be included in the evaluation of food-grade carrageenan. Poligeenan (average molecular weight 10-20 kDa) has not been authorised as a food additive and is not used in any food applications. In its evaluation of carrageenan (E 407) and processed Eucheuma seaweed (E 407a), the Panel noted that the ADME database was sufficient to conclude that carrageenan was not absorbed intact; in a subchronic toxicity study performed with carrageenan almost complying with the EU specification for E 407 in rats, the no-observed-adverse-effect level (NOAEL) was 3,400-3,900 mg/kg body weight (bw) per day, the highest dose tested; no adverse effects have been detected in chronic toxicity studies with carrageenan in rats up to 7,500 mg/kg bw per day, the highest dose tested; there was no concern with respect to the carcinogenicity of carrageenan; carrageenan and processed Eucheuma seaweed did not raise a concern with respect to genotoxicity; the NOAEL of sodium and calcium carrageenan for prenatal developmental dietary toxicity studies were the highest dose tested; the safety of processed Eucheuma seaweed was sufficiently covered by the toxicological evaluation of carrageenan; data were adequate for a refined exposure assessment for 41 out of 79 food categories. However, the Panel noted uncertainties as regards the chemistry, the exposure assessment and biological and toxicological data. Overall, taking into account the lack of adequate data to address these uncertainties, the Panel concluded that the existing group acceptable daily intake (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.