Scientific opinion on Prosmoke BW 01.

The EFSA Panel on Food Additives and Flavourings (FAF) was requested to evaluate the safety of Prosmoke BW 01 as a new smoke flavouring primary product, in accordance with Regulation (EC) No 2065/2003. Prosmoke BW01 is produced by pyrolysis of beechwood (Fagus sylvatica L.) sawdust. Its water content is estimated at 56 wt%, the total identified volatile fraction accounts for 28 wt% of the primary product, corresponding to 64% of the solvent-free mass, while the unidentified fraction amounts to 16 wt% of the primary product. Analytical data provided for three batches demonstrated that their batch-to-batch-variability was sufficiently low. However, for the batch used for the toxicological studies, there were substantial deviations in the concentration of nearly all the constituents compared to the other three batches. The dietary exposure of Prosmoke BW 01 was estimated to be between 6.2 and 9.2 mg/kg body weight (bw) per day, respectively, using SMK-EPIC and SMK-TAMDI. Using the FAIM tool, the 95th percentile exposure estimates ranged from 3.2 mg/kg bw per day for the elderly to 17.9 mg/kg bw per day for children. The Panel noted that furan-2(5H)-one is present in all batches of the primary product at an average concentration of 0.88 wt%. This substance was evaluated by the FAF Panel as genotoxic in vivo after oral exposure. The Panel considered that the (geno)toxicity studies available on the whole mixture were not adequate to support the safety assessment, due to limitations in these studies and because they were performed with a batch which may not be representative for the material of commerce. Considering that the exposure estimates for furan-2(5H)-one are above the TTC value of 0.0025 µg/kg bw per day (or 0.15 µg/person per day) for DNA-reactive mutagens and/or carcinogens, the Panel concluded that Prosmoke BW 01 raises a concern with respect to genotoxicity.

Safety of ethyl acrylate to be used as flavouring.

The EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF Panel) was requested by the European Commission according to Art. 29 1(a) of the Regulation (EC) No 178/2002 to carry out a review of existing literature on the safety of ethyl acrylate [FL-no: 09.037] when used as a flavouring substance. Ethyl acrylate [FL-no: 09.037] was evaluated in 2010 by EFSA in FGE.71 as a flavouring substance, based on the 2006 JECFA evaluation. The Panel concluded that ethyl acrylate was of no safety concern at estimated level of intake as flavouring substance based on the Maximised Survey-Derived Daily Intake (MSDI) approach. The Panel has evaluated the new literature available and any previous assessments performed by JECFA (2006) and EFSA (2010). Moreover, new data on the use levels of ethyl acrylate as flavouring substance have been provided. For use as flavouring substance, the chronic dietary exposure estimated using the added portions exposure technique (APET), is calculated to be 3,545 µg/person per day for a 60-kg adult and 2,233 µg/person per day for a 15-kg 3-year-old child. Exposure from food contact materials may be up to 6,000 µg/person per day. The Panel considered that based on the available data, which covers all relevant genetic endpoints (i.e. gene mutations, structural and numerical chromosomal aberrations) there is no concern with respect to genotoxicity of ethyl acrylate. The Panel evaluated the available carcinogenicity studies conducted in rats and mice and agreed with the NTP evaluation (1998) concluding that the forestomach squamous cell papilloma and carcinoma observed in rodents were not relevant to humans. Additionally, there was no evidence of systemic toxicity in short-term and subchronic toxicity studies. Therefore, the Panel concluded that there is no safety concern for the use of ethyl acrylate as a flavouring substance, under the intended conditions of use.

Safety of benzophenone to be used as flavouring.

Benzophenone [FL-no: 07.032] has been evaluated as a flavouring substance, in FGE.69, by the EFSA Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food in 2008. Benzophenone was evaluated also by JECFA (2011) and by IARC (2013) based on studies that were not considered in the EFSA opinion on FGE.69. Therefore, the Commission requested the CEF Panel to carry out a review of existing literature on the safety of this flavouring substance. In the framework of the evaluation of benzophenone as a food contact material, the CEF Panel established a tolerable daily intake (TDI) of 0.03 mg/kg body weight (bw) per day (2009). In the present Opinion, the Panel considered the already existing evaluations by EFSA, JECFA, IARC and available literature data on benzophenone toxicity. Moreover, new data on the use levels of benzophenone as a flavouring substance have been provided. The Panel considers that there is no concern with respect to genotoxicity. The Panel considers the endocrine activities of benzophenone and its metabolite 4-hydroxybenzophenone as weak and not directly related to the observed toxic effects including the neoplastic effects in rodents. The Panel confirms that the conservative approach taken by EFSA (2009) to derive a TDI of 0.03 mg/kg bw for benzophenone is appropriate to cover the non-neoplastic effects in the chronic toxicity studies and the neoplastic effects induced in the rodent carcinogenicity studies. The TDI is in the same order of magnitude as the chronic dietary exposure of adults and children to benzophenone (10-20 µg/kg bw per day) for the amount of added flavouring substance. The Panel considers that the calculated TDI and exposure estimate are based on conservative assumptions. The Panel concludes that there is no safety concern for benzophenone under the current condition of use as a flavouring substance.

Scientific Opinion of Flavouring Group Evaluation 500 (FGE.500): rum ether.

The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids of the European Food Safety Authority was requested to deliver a scientific opinion on the implications for human health of the flavouring rum ether [FL-no: 21.001] in the Flavouring Group Evaluation 500 (FGE.500), according to Regulation (EC) No 1331/2008 and Regulation (EC) No 1334/2008 of the European Parliament and of the Council. Rum ether is a complex mixture of volatile substances obtained by distillation of the reaction products of pyroligneous acid and ethyl alcohol under oxidative conditions in the presence of manganese dioxide and sulfuric acid. A total of 84 volatile constituents have been reported by the applicant. It is a colourless liquid with a rum-like odour and flavour. Its major uses are in the food categories beverages, confectionery and baked goods. The Panel decided to apply a congeneric group-based approach. The 84 reported constituents were allocated to 12 congeneric groups, based on structural and metabolic similarity. For eight of the congeneric groups, the Panel concluded that there is no safety concern at the intended conditions of use. However, the Panel concluded that substances in congeneric group 1 (ethanol and acetaldehyde) and congeneric group 12 (furan) are carcinogenic and genotoxic. The Panel also identified genotoxicity concerns for substances in congeneric group 3 (3-pentene-2-one). The exposure for congeneric group 10 (ethers of various structures) was above the Threshold of Toxicological Concern (TTC) applicable for this group, but a point of departure or health based guidance value that covers all the substances in this group could not be identified. The Panel concluded that according to the overall strategy for the risk assessment of flavouring substances, the presence of genotoxic substances as process-derived constituents of rum ether is of safety concern.

The food processing contaminant glyoxal promotes tumour growth in the multiple intestinal neoplasia (Min) mouse model.

Glyoxal is formed endogenously and at a higher rate in the case of hyperglycemia. Glyoxal is also a food processing contaminant and has been shown to be mutagenic and genotoxic in vitro. The tumourigenic potential of glyoxal was investigated using the multiple intestinal neoplasia (Min) mouse model, which spontaneously develops intestinal tumours and is susceptible to intestinal carcinogens. C57BL/6J females were mated with Min males. Four days after mating and throughout gestation and lactation, the pregnant dams were exposed to glyoxal through drinking water (0.0125%, 0.025%, 0.05%, 0.1%) or regular tap water. Female and male offspring were housed separately from PND21 and continued with the same treatment. One group were only exposed to 0.1% glyoxal from postnatal day (PND) 21. There was no difference in the number of intestinal tumours between control and treatment groups. However, exposure to 0.1% glyoxal starting in utero and at PND21 caused a significant increase in tumour size in the small intestine for male and female mice in comparison with respective control groups. This study suggests that glyoxal has tumour growth promoting properties in the small intestine in Min mice.

Intestinal Tumor Development in C57BL/6J-ApcMin/+ Mice Expressing Human Sulphotransferases 1A1 and 1A2 After Oral Exposure to 2,5-Dimethylfuran.

BACKGROUND: 2,5-dimethylfuran (DMF) is formed during heating of foods. Following side chain hydroxylation, DMF could be a substrate for human sulphotransferases (SULTs), which may lead to formation of a DNA reactive electrophile. Only few conflicting in vitro and no in vivo studies on DMF currently exist. MATERIALS AND METHODS: The tumorigenic potential of DMF was studied in multiple intestinal neoplasia Apc(Min/+) (Min) mice that are sensitive to intestinal carcinogenesis and express hSULTs 1A1 and 1A2 (Min/hSULT). Min and Min/hSULT mice were orally exposed to DMF for six weeks. RESULTS: The intestinal tumor development of untreated female Min/hSULT mice was significantly lower compared to that of untreated Min females. No such effects of hSULTs were seen in males. DMF had a weak tumorigenic potential in the colon of female Min/hSULT mice, but not in males. Tumor development in Min mice was not affected. CONCLUSION: Overall, the tumorigenic potential of DMF in a metabolically competent mouse model was not convincing.

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.

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.

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.

The min mouse on FVB background: susceptibility to spontaneous and carcinogen-induced intestinal tumourigenesis.

The multiple intestinal neoplasia (Min) mouse is a model for intestinal tumourigenesis. On the C57BL/6J (B6) background, with an incidence of 100%, the Min mouse develops numerous tumours throughout the intestine, particularly in the small intestine. The Min phenotype was backcrossed to the FVB/NJ (FVB) genetic background in order to reduce the number of tumours. Control FVB Min mice had an incidence of 7% tumours, both in the small intestine and in the colon. One or more flat aberrant crypt foci (ACF) were also observed in the colon of 20% of the control mice. Neonatal mice were given one dose of the food-processing contaminant 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in order to test the chemical induction of tumours and flat ACF. Treatment with PhIP significantly increased both the number and incidence of tumours in the small intestine, and gave a non-significant increase of tumours and flat ACF in the colon.

Retinol-induced intestinal tumorigenesis in Min/+ mice and importance of vitamin D status.

The effects of life-long dietary exposure, starting in utero, to high retinol, low vitamin D, or high retinol in combination with low vitamin D on intestinal tumorigenesis in Min/+ mice were investigated. In males, high retinol alone significantly increased the number (2.6-fold) and size (1.3-fold) of small intestinal tumours; in females no significant increase in tumour number or size was seen. In both genders, low vitamin D intake alone did not affect intestinal tumorigenesis. In males, intake of the combined high retinol/low vitamin D diet did not further increase the effects caused by high retinol alone. In females, however, the high retinol/low vitamin D-induced increase in tumour number (3.1-fold) and tumour size (1.5-fold) exceeded that of high retinol alone. In conclusion, a high dietary intake of retinol stimulated intestinal tumorigenesis in Min/+ mice. Furthermore, the results indicate a combined effect of high retinol and low vitamin D on tumorigenesis in females.

5-Hydroxymethylfurfural and 5-sulfooxymethylfurfural increase adenoma and flat ACF number in the intestine of Min/+ mice.

BACKGROUND: 5-Hydroxymethylfurfural (HMF) is produced in large quantities during the processing of food containing carbohydrates and can be metabolised to 5-sulfooxymethylfurfural (SMF), a reactive intermediate that can bind to DNA and cause mutagenic effects. MATERIALS AND METHODS: Three to six days after birth, multiple intestinal neoplasia (Min/+) mice were given a single subcutaneous injection of either 500 mg/kg body weight (bw) HMF, 25 mg/kg bw SMF or vehicle (0.9 % NaCl), and were euthanised at 12 weeks of age. The number and size of adenomas and flat aberrant crypt foci (ACF) were counted in the intestine. RESULTS: HMF increased the number of small intestinal adenomas (p=0.033), whereas SMF increased the flat ACF number in the large intestine (p=0.025). Treatment with HMF and SMF had no effect on the size of the adenomas. CONCLUSION: These results show that both HMF and SMF are weak intestinal carcinogens in Min/+ mice.