Camilla Svendsen

Intestinal carcinogenesis of two food processing contaminants, 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine and 5‐hydroxymethylfurfural, in transgenic FVB min mice expressing human sulfotransferases

Humans express sulfotransferases (SULTs) of the SULT1A subfamily in many tissues, whilst the single SULT1A gene present in rodents is mainly expressed in liver. The food processing contaminants, 5‐hydroxymethylfurfural (HMF) and 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), are bioactivated by human SULT1A1 and SULT1A2. FVB multiple intestinal neoplasia (Min) mice, which spontaneously develop tumors and flat aberrant crypt foci (ACF) in intestine, were crossed with transgenic FVB mice expressing human SULT1A1 and 1A2 (hSULT) in several tissues, giving rise to wild‐type and Min mice with and without hSULT. One‐week‐old Min mice with or without hSULT were given HMF (375 or 750 mg/kg bw) or saline by gavage three times a week for 11 wk. In another experiment, the F1 generation received subcutaneous injections of 50 mg/kg bw PhIP or saline 1 wk before birth, and 1, 2, and 3 wk after birth. HMF did not affect the formation of tumors, but may have induced some flat ACF (incidence 15–20%) in Min mice with and without hSULT. No control mouse developed any flat ACF. With the limitation that these putative effects were weak, they were unaffected by hSULT expression. The carcinogenic effect of PhIP increased in the presence of hSULT, with a significant increase in both incidence (31–80%) and number of colonic tumors (0.4–1.3 per animal). Thus, intestinal expression of human SULT1A1 and 1A2 might increase the susceptibility to compounds bioactivated via this pathway implying that humans might be more susceptible than conventional rodent models.

Genotoxicity of three food processing contaminants in transgenic mice expressing human sulfotransferases 1A1 and 1A2 as assessed by the in vivo alkaline single cell gel electrophoresis assay

The food processing contaminants 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), 5‐hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N‐hydroxy‐PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild‐type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N‐hydroxy‐PhIP and HMF in vivo. Environ. Mol. Mutagen. 56:709–714, 2015.

The impact of commercial rodent diets on the induction of tumours and flat aberrant crypt foci in the intestine of multiple intestinal neoplasia mice

A large variation in spontaneous tumour development in the multiple intestinal neoplasia (Min) mouse model between laboratories has been reported. The composition of the diet might be an important factor. We examined the impact of five commercial rodent diets: the natural ingredient breeding diet Harlan Teklad 2018 (HT), the purified breeding diet AIN93G, the natural ingredient maintenance diet RM1, and the purified maintenance diets AIN93M and AIN76A, on the spontaneous intestinal tumorigenesis in the Min mouse model. The Min mice were fed one of two breeding diets during gestation and until four weeks of age, thereafter one of the three maintenance diets. Min mice bred on the breeding diet HT had significantly higher numbers and incidences of tumours in the colon, but fewer tumours in the small intestine than the breeding diet AIN93G. The maintenance diet RM1 gave a significantly higher number of small intestinal and colonic tumours and precancerous lesions called flat aberrant crypt foci (ACF) compared with the maintenance diets AIN93M and AIN76A. These findings show the importance of defining the type of diet used in experimental intestinal carcinogenesis studies, and that the diet should be taken into consideration when comparing results from different studies with Min mice.

Scientific Opinion of Flavouring Group Evaluation 410 (FGE.410): 4’,5,7‐trihydroxyflavanone from chemical group 25 (phenol derivatives containing ring‐alkyl, ring‐alkoxy, and side‐chains with an oxygenated functional group)

Abstract The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) of EFSA was requested to deliver a scientific opinion on the implications for human health of the flavouring substance 4′,5,7‐trihydroxyflavanone or naringenin [FL‐no: 16.132], in the Flavouring Group Evaluation 410 (FGE.410), according to Regulation (EC) No 1331/2008 of the European Parliament and of the Council. The substance occurs naturally in grapefruits, oranges and tomatoes. It is intended to be used as a flavouring substance with flavour‐modifying properties in specific categories of food. Information on specifications and manufacturing of [FL‐no: 16.132] were considered adequate; however, data on stability in food are incomplete. The Panel noted that the available genotoxicity studies have significant shortcomings and are insufficient to conclude on the genotoxic potential of naringenin. Therefore, [FL‐no: 16.132] cannot be evaluated through the Procedure. Additionally, the Panel noted that inhibition of CYP 450 by [FL‐no: 16.132] has been clearly demonstrated in animal species in vivo which implies that the substance may interact with the metabolism and elimination of medicines and no convincing information is available that this does not pose a risk to humans at the estimated levels of exposure. To continue with the safety assessment of [FL‐no: 16.132], a bacterial gene mutation assay and an in vitro micronucleus assay (according to OECD guidelines 471, 487 and GLP) are required. Even if these studies do not indicate a genotoxic potential, additional toxicological data are needed to finalise the evaluation.

An in vitro study on the genotoxic effect of substituted furans in cells transfected with human metabolizing enzymes: 2,5-dimethylfuran and furfuryl alcohol

2,5-Dimethylfuran (DMF) and furfuryl alcohol (FFA) are two substituted furans that are formed during the processing of foods and have also been used as food flavorings. DMF and FFA are proposed to be bioactivated by human sulfotransferases (SULTs) which are not expressed in conventional cell lines used for genotoxicity testing. Therefore, in addition to the standard V79 cell line, we used a transfected V79 derived cell line co-expressing human cytochrome P450 (CYP) 2E1 and human SULT1A1 to assess the genotoxicity of DMF and FFA. The alkaline single cell gel electrophoresis (SCGE) assay was used to detect DNA damage in the form of single strand breaks and alkali-labile sites after exposure to DMF (0.5h; 0.5, 1, 1.5 or 2mM) or FFA (3h; 1, 3, 6 or 15mM). DMF induced DNA damage in V79 cells in a concentration-dependent manner irrespective of the expression of human CYP2E1 and SULT1A1. Almost no increase in the level of DNA damage was detected after exposure to FFA, except for a weak effect at the highest concentration in the transfected cell line. The results suggest that DNA damage in V79 cells from exposure to DMF detected by the alkaline SCGE assay is independent of human CYP2E1 and SULT1A1, and the genotoxic effect of FFA, as assessed by SCGE, is minimal in V79 cells.

Hypotheses and evidence related to intense sweeteners and effects on appetite and body weight changes: A scoping review of reviews

Observed associations between consumption of diet foods and obesity have sparked controversy over whether intense sweeteners may promote weight gain, despite their negligible energy contribution. We conducted a scoping review of reviews, to obtain an overview of hypotheses, research approaches and features of the evidence on intense sweeteners’ potential relationships to appetite and weight changes. We searched for reviews of the scientific literature published from 2006 to May 2017. Two reviewers independently assessed title and abstracts, and full text publications. Arksey and O’Malley’s framework for scoping reviews guided the process. We extracted and charted data on characteristics of the reviews and the evidence presented. The 40 included reviews present hypotheses both on how intense sweeteners can reduce or maintain body weight and on how these can promote weight gain. We classified only five publications as systematic reviews; another nine presented some systematic approaches, while 26 reviews did not describe criteria for selecting or assessing the primary studies. Evidence was often presented for intense sweeteners as a group or unspecified, and against several comparators (e.g. sugar, water, placebo, intake levels) with limited discussion on the interpretation of different combinations. Apart from the observational studies, the presented primary evidence in humans is dominated by small studies with short follow-up—considered insufficient to assess weight change. Systematic reviews of animal studies are lacking in this topic area. The systematic evidence only partly explore forwarded hypotheses found in the literature. Primary studies in humans seem to be available for systematic exploration of some hypotheses, but long-term experimental studies in humans appear sparse. With few exceptions, the reviews on intense sweeteners and weight change underuse systematic methodology, and thus, the available evidence. Further studies and systematic reviews should be explicit about the hypothesis explored and elucidate possible underlying mechanisms.

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

Abstract 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.

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

Abstract 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.

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)

Abstract 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 of Flavouring Group Evaluation 502 (FGE.502): grill flavour ‘Grillin’ 5078’

Abstract The EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF Panel) was requested to deliver a scientific opinion on the implication for human health of the product Grillin’ 5078 [FL‐no: 21.003] in the Flavouring Group Evaluation 502, according to Regulation (EC) No 1331/2008 and Regulation (EC) No 1334/2008 of the European Parliament and of the Council. The product is derived from heat‐treated high oleic sunflower oil and intended to be used as a food flavouring with charbroiled or grilled aroma in a wide variety of food categories either in liquid or powder form. Information on manufacturing and compositional data was considered adequate to show the reproducibility of the production process. However, the Panel noted that a considerable amount of the non‐volatile fraction of the product could not be identified. The chronic dietary exposure to the substance estimated using the Added Portions Exposure Technique (APET) was calculated to be 60 mg/person per day for a 60‐kg adult and 37.8 mg/person per day for a 15‐kg child. The data submitted for evaluating the genotoxic potential of the flavouring was considered insufficient. There are still 12 substances in the flavouring for which the evaluation of genotoxic potential is pending. No toxicity studies have been provided on the final product itself. Only information on a number of constituents of the flavouring and data on toxicity of several thermally treated fats and oils were provided by the applicant. However, the Panel considered the time–temperature conditions that were applied in the preparation of the substances tested as not comparable to those applied in the course of the production of the flavouring. The Panel concluded that on the basis of the data provided by the applicant the safety of Grillin’ 5078 cannot be established.