Tiemann Ute

Brüssow Klaus-Peter

Sven Dänicke

Vanselow Jens

J Vanselow
email: vanselow@fbn-23dummerstorf.de

Feeding of pregnant sows with mycotoxin contaminated diets has no effect 1 on foetal and maternal hepatic transcription of genes of the insulin-like 2 growth factor system 3 4 5 6

Keywords: Deoxynivalenol, Zearalenone, IGF-system, foetus, neonatal pig, liver 44 45

Feeding of pregnant sows with mycotoxin contaminated diets has no effect on fetal and maternal hepatic transcription of genes

hepatic levels of the IGF system. We aimed to determine (1) the normal foetal and maternal 28 hepatic transcription of major IGF genes at distinct stages of pregnancy and to find out, (2) if 29 the mycotoxins deoxynivalenol (DON) and zearalenone (ZON) affect transcript 30 concentrations. Pregnant sows were fed with naturally contaminated diets from days 35 to 70 31 (experiment 1), or from days 75 to 110 (experiment 2), or with control diets. Foetal hepatic 32 IGF transcripts were determined at days 70 and 110, maternal transcripts at day 70 by qPCR. 33

Highest levels of IGF-I transcripts were found in sows. Contrary, IGF-II was predominantly 34 expressed in foetuses at day 70. Expression of IGF-IR and IGFBP-3 decreased, whereas that 35 of IGFBP-2 increased towards term. IGF-IIR and IGFBP-1 expression was similar in both, 36 foetuses and full-term piglets. IGF-IIR showed reduced levels in the maternal liver. Exposure 37 of pregnant sows to DON and ZON contaminated diets significantly reduced the maternal and 38 tended to reduce the weight gain of piglets in experiment 2 but had no effects on hepatic 39 levels of IGF transcripts in both experiments. This suggests that mycotoxin contaminated 40 diets can induce growth depression in pigs during pregnancy without affecting hepatic 41 transcription of major IGF genes. 42

Introduction 47

Mycotoxins are biologically active secondary fungal metabolites found as contaminants of 48 feedstuffs, which exert toxic effects on animals and human beings [START_REF] Fink-Gremmels | Mycotoxins: their implications for human and animal health[END_REF]. 49 Among farm animals, pigs are most susceptible to deoxynivalenol (DON), resulting in 50 guidance values for critical dietary concentrations of 0.9 mg DON/kg for all pig categories; of 51 0.1 mg zearalenone (ZON)/kg for piglets and gilts, and of 0.25 mg ZON/kg for sows and 52 fattening pigs (The Commission of the European Communities 2006). The presence of DON 53 in swine feedstuffs decreases feed intake, causes feed refusal, and induces occasional 54 vomiting [START_REF] Conkova | Fusarial toxins and their role in 322 animal diseases[END_REF][START_REF] Dänicke | Fusariumtoxine in der Tierernährung[END_REF][START_REF] Diekman | Mycotoxins and reproduction in domestic livestock[END_REF]. Besides these 55 effects, reproductive alterations have been observed in humans and pigs [START_REF] Alm | 311 Influence of Fusarium-toxin contaminated feed on initial quality and meiotic competence of 312 gilt oocytes[END_REF]56 Hussein and Brasel 2001;Tiemann and Dänicke 2007). 57 The data reported here were collected in the context of a large study analyzing the transfer of 59 DON and ZON from sows to their foetuses during pregnancy and their influence on 60 physiological parameters in sows and foetuses/piglets [START_REF] Dänicke | On the 333 transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from the sow 334 to the full-term piglet during the last third of gestation[END_REF]Goyarts et al. 61 2007;[START_REF] Tiemann | Changes in the spleen and liver of pregnant sows and full-term piglets after 455 feeding diets naturally contaminated with deoxynivalenol and zearalenone[END_REF]. Feeding pregnant sows with a Fusarium toxin contaminated 62 triticale diet (4.42 mg DON and 0.048 mg ZON per kg diet) in the period from day 35 to 70 of 63 gestation, DON, ZON and their metabolites were detected foetally on day 70 (Goyarts et al. 64 2007), but neither the live weight of sows nor the foetus weight was significantly reduced. 65 Furthermore, no teratogenic or embryolethal effects could be observed in experimental 66 groups. Histopathological evaluation of tissues from sows of the treated group revealed 67 changes in liver and spleen tissues, whereas no significant changes were observed in these 68 tissues in their piglets [START_REF] Tiemann | Changes in the spleen and liver of pregnant sows and full-term piglets after 455 feeding diets naturally contaminated with deoxynivalenol and zearalenone[END_REF]). However, pregnant sows which were fed a 69 Fusarium toxin contaminated diet containing 9.57 mg DON and 0.36 mg ZON per kg 70 between the gestation days 75 to 110, reduced their mean feed intake. As a result of the initial 71 maternal feed intake depression, the mean live weight of foetuses and the live weight gain in 72 mothers was reduced. However, macroscopic lesion in any organs of sows and piglets were 73 not observed [START_REF] Dänicke | On the 333 transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from the sow 334 to the full-term piglet during the last third of gestation[END_REF]. 74

75

The IGF axis is highly responsive to the nutritional status [START_REF] Ketelslegers | Nutritional 387 regulation of insulin-like growth factor-I[END_REF]Simmen et 76 al. 1998;[START_REF] Thissen | Nutritional regulation of the insulin-449 like growth factors[END_REF]. In sheep, reduced growth correlates with lower IGF-I 77 expression in the liver, but not in skeletal muscles, where no changes occurred (Pell et al. 78 1993). The liver is generally thought to be the major production site for IGFs (Jones and 79 Clemmons 1995;[START_REF] Ohlsson | The relative importance of 418 endocrine versus autocrine/paracrine insulin-like growth factor-I in the regulation of body 419 growth[END_REF]. In adult rats, IGF-I transcripts were found in many 80 tissues, but had highest concentrations in the liver [START_REF] Murphy | Tissue distribution of insulin-like growth factor I 416 and II messenger ribonucleic acid in the adult rat[END_REF]. The IGF-system is 81 important in regulating foetal and placental growth [START_REF] Dechiara | A growth-deficiency phenotype in 339 heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting[END_REF][START_REF] Devrijer | Altered 347 placental and foetal expression of IGFs and IGF-binding proteins associated with intrauterine 348 growth restriction in foetal sheep during early and mid-pregnancy[END_REF]82 Han and Hill 1992;[START_REF] Liu | Insulin-like growth factors (IGFs) and 403 IGF-binding proteins in the developing rhesus monkey[END_REF]. It acts via endocrine as well as via autocrine/paracrine 83 mechanisms and has metabolic and anabolic functions [START_REF] Ashworth | Causes and 314 consequences of foetal growth retardation in pigs[END_REF]Jones and 84 Clemmons 1995). Functionally, the IGF-system comprises the ligands, IGF-I and -II, their 85 receptors, and the IGF-binding proteins (IGFBP). The various IGFBPs are modulators of the 86 biological activity of IGFs and each IGFBP can act in either a paracrine or autocrine manner 87 to elicit its own set of biological responses [START_REF] Mccusker | The insulin-like growth factor binding proteins: 408 structure and biological functions[END_REF]. 88

During the present study, firstly, the hepatic expression of major genes of the IGF-system 90 during pregnancy as reflected by transcripts for IGF-I, IGF-II, their receptors IGF-IR and 91

Materials and methods 98

Experimental design and diets 99

Experiments were performed with pregnant gilts (German Landrace). Sows were kept 100 individually without bedding on a partially slatted floor. Water and food was provided ad 101 libitum. Animals were fed on a wheat diet, blended with batches of triticale or wheat that were 102 partly naturally contaminated with Fusarium toxins. In experiment 1, the contaminated diet 103 contained 15% of Fusarium toxin contaminated triticale which resulted in a dietary DON and 104 ZON concentration of 4.42 and 0.048 mg/kg, respectively (Tab. 1). This diet was fed from 105 days 35 to 70 of pregnancy. In experiment 2, 40% of a Fusarium toxin contaminated wheat 106 was included in the diet of the experimental group which resulted in dietary DON and ZON 107 concentrations of 9.57 and 0.358 mg/kg, respectively (Tab. 1). This diet was fed from days 75 108 to 110 of pregnancy. The diet for the control groups in both experiments also contained 15% 109 and 40% of triticale and wheat, respectively. However, the DON and ZON concentrations of 110 control diets were 0.15 and 0.004 mg/kg, respectively, in experiment 1, and 0.210 and 0.004 111 mg/kg diet in experiment 2. Both, according to diet composition (commonly used feedstuffs) 112 and because of the very low toxin levels, these control diets are in the range of commonly 113 used complete diets for pigs [START_REF] Meng | Undesirable substances in animal 411 nutrition -minimising is possible[END_REF]. Analysis of wheat and triticale by GC-MS for 114 a detailed characterization of the trichothecene mycotoxin pattern revealed that further B-115 trichothecene mycotoxins such as 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol and 116 nivalenol, and of the A-trichothecene mycotoxin HT-2 toxin were all lower than the detection 117 limits. 118 119 Detailed information on the composition of the diet was also described by Goyarts et al. 120 (2007) for experiment 1 and by [START_REF] Dänicke | On the 333 transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from the sow 334 to the full-term piglet during the last third of gestation[END_REF] Total RNA was prepared with the RNeasy mini kit (Qiagen, Hilden, Germany). Briefly, tissue 152 chunks, previously conserved in RNAlater (Qiagen), were transferred into a guanidine iso-153 thiocyanate containing buffer and homogenized with an Ultra Turrax (IKA instruments, 154

Germany). Subsequently one volume of 70% ethanol (v/v) was added to the homogenized 155 lysates and the RNA was extracted from the samples by adsorption to silica gel spin columns. 156

After three washing steps and elution in 30 µl deionised, RNAse free water (washing buffers 157 and water provided by the kit), RNA was quantified in a GeneQuant II instrument 158 (Pharmacia, Freiburg, Germany). Quality of RNA was monitored from randomly selected 159 LightCycler-FastStart DNA Master PLUS SYBR Green I Kit (Roche) in 10µl total reaction 172 volume. Amplification and quantification of products were performed in a LightCycler® I 173 instrument (Roche) under the following cycling conditions: Pre-incubation at 95°C for 10´, 174 followed by 45 cycles denaturation at 95°C for 15´´, annealing at 60°C for 10´´, extension at 175 72°C for 10´´ and single point fluorescence acquisition at 83°C for 6´´. The melting peaks of 176 all samples were routinely determined by melting curve analysis in order to ascertain that only 177 the expected products had been generated. Additionally, the length of all PCR products was 178 monitored by agarose gel electrophoresis analysis (3% agarose, ethidium bromide stained). 179

Cloned PCR products of the respective genes were used to generate external standard curves. 180

Routinely, dilutions of standards covering five orders of magnitude (5 x 10 -16 to 5 x 10 -12 g 181 DNA/reaction) were freshly diluted from stocks of 10ng DNA/µl and co-amplified during 182 each run. Copy numbers were calculated relative to the amount of total RNA previously 183 subjected to cDNA synthesis. To normalize for variations between individual LightCycler 184 runs one or two arbitrarily selected samples were co-amplified as calibrators. 185 186

Statistical analysis 187

The data were analyzed using the Student-Newman-Keuls test following one-way analysis of 188 variance (ANOVA) using the SigmaStat Statistical Analysis System (Jandel Scientific, San 189

Hepatic transcripts of IGF genes show very different levels during ontogenesis 196

Hepatic transcript levels of different IGF genes were found at very different expression levels 197 covering several orders of magnitude. IGF-II transcripts exceeded levels of 4x10 7 copies/µg 198 RNA in foetal livers at day 70, whereas foetal expression of IGF-I was more than three orders 199 of magnitude lower (Fig. 1). Most of the transcripts, IGF-II, IGF-IR, 200 showed highest hepatic concentrations during foetal life. However, two of the transcripts 201 showed an opposite ontogenetic regulation. Particularly, levels of IGF-I transcripts in livers of 202 pregnant sows, exceeded foetal levels by 14 to 17 fold. But also IGFBP-3 showed 203 significantly increased levels in adult animals. The levels of some transcripts significantly 204 decreased during foetal development as observed in case of IGF-II, IGF-IR, and IGFBP-3. 205 However, IGFBP-2 transcript concentrations significantly increased towards term. Transcript 206 levels of IGF-I and IGF-IIR did not show significant differences between both foetal stages 207 but had significantly higher or lower levels in adult sows, respectively. IGFBP-1 transcripts 208

showed similar levels at all ontogenetic stages but tended to slightly decrease with age. 209 210

Hepatic transcript levels are not affected by diets naturally contaminated with mycotoxins 211

In experiment 1 no differences in feed intake were observed between the experimental and 212 control groups. The live weight gain over the tested gestation period between day 35 and 70 213 was not significantly influenced by feeding the mycotoxin contaminated diet. The mean fetus 214 weight was not significantly different between the control group and the group fed with a 215 Fusarium toxin contaminated diet [START_REF] Goyarts | On the 374 transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from sows to 375 their fetuses during days 35-70 of gestation[END_REF]. 216 217 In experiment 2, the sows fed with the Fusarium toxin contaminated diet consumed 38% less 218 feed than the sows fed with the control diet and the live weight over the tested gestation 219 period between day 75 and 110 was significantly reduced by 93%. The mean weight of piglets 220 also tended to be reduced from 1335.2 ± 347.0 g to 1120.9 ± 260.2 g (p=0.087) in the control 221 and experimental group, respectively. The liver weight was not altered in sows. At terminal 222 necropsy there was no macroscopic lesion observed in any organ of sows and piglets exposed 223 to the Fusarium toxin [START_REF] Dänicke | On the 333 transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from the sow 334 to the full-term piglet during the last third of gestation[END_REF]. 224 225The hepatic transcript concentrations of all IGF genes however were not significantly affected 226 by feeding pregnant sows for 35 days at different stages of gestation with diets naturally 227

  in maternal livers at day 70 of gestation. Secondly, to find out if the hepatic 93 expression of the IGF system is affected by mycotoxin contaminated diets, sows were fed 94 with diets naturally contaminated with Fusarium toxins during days 35 to 70 and 75 to 110 of 95 pregnancy and the respective levels of IGF transcripts were determined. 96 97

  for experiment 2. The experiments were 121 terminated by delivering the foetuses by Caesarean section. Liver samples from foetuses 122 (n=10)/full-term piglets (n=10) were collected on days 70 and 110, from sows (n=5) only on 123 days 70 of pregnancy. The samples were cut into pieces of about 0.5 cm diameter, 124 immediately transferred to RNAlater solution (Qiagen, Hilden, Germany), incubated for 24 125 diets was analyzed by HPLC with DAD (diode array detection) after a clean-129 up with IAC (immunoaffinity column, DONprep™, R-Biopharm Rhone Ltd.,Darmstadt, 130 Germany) according to a slightly modified procedure of R-Biopharm Rhone. The detection 131 limit was 0.03 mg/kg and the recovery approximately 89%. Further trichothecenes in wheat 132 and triticale were analysed by the Institute of Animal Nutrition of the University of 133 Hohenheim, Germany, using a GC-MS method[START_REF] Dänicke | On the effects of graded levels of Fusarium toxin contaminated wheat in diets for gilts 330 on feed intake, growth performance and metabolism of deoxynivalenol and zearalenone. 331[END_REF]. 134 135Caesarean section and collection of physiological samples 136Both experiments were terminated after exposure to the experimental and control diets for 35 137 days. At both time points, pregnant sows were anaesthetized by an intravenous injection with 138 ketamin/xylazin (15 mg/kg Ursotamin® and 1.5 mg/kg Xylazin ® , Serumwerk Bernburg, 139 Germany). Following anaesthesia, the fetuses/full-term piglets were delivered by Caesarean 140 section. The fetuses/full-term piglets where euthanized after cutting the umbilical cord by ) via the umbilical vein. Fetuses/full-term piglets were weighed 143 and the head-rump-length was measured. Thereafter, livers were dissected and weighed. After 144 euthanizing the sows with T61, livers were dissected and weighed too. 145 146 The present study was approved by the Committee for Animal Use and Care of the 147 Ministerial Agricultural Department of Mecklenburg-Vorpommern, Germany, according to 148 the German Law for Animal Protection (TierSchG). 149 150 RNA preparation, cDNA synthesis and quantitative PCR (qPCR) 151

  primers for cDNA synthesis and amplification of porcine IGF-I, IGF-II, IGF-162 IR, IGF-IIR, and IGFBP-1, 2, 3 transcripts were designed according to published mRNA 163 sequences (Tab. 2). For cDNA synthesis, 0.5 µg total RNA was reversely transcribed in a 164 25µl reaction volume using M-MLV reverse transcriptase, RNase H Minus, Point Mutant 165 (Promega, Mannheim, Germany) with gene-specific primers. The freshly synthesized cDNA 166 samples were cleaned with the High Pure PCR Product Purification Kit (Roche, Mannheim, 167 Germany) and eluted in 50 µl elution buffer. The identity of products generated with different 168 primer pairs was controlled by sequencing. 169 170 For qPCR, routinely, 0.25 and 0.5µl of the cDNA samples were amplified with the 171

  Rafael, CA). The data represent means ± SEM. All results with P<0.05 were considered 190 statistically significant. The number of animals used in both of the experiments is described in

Figure 1 :Figure 2 :

 12 Figure captions 460 461 Figure 1: Normal hepatic transcript concentrations of genes of the IGF system in fetuses/full-462 term piglets at days 70 and 110 and sows at day 70 of gestation. The sows were fed with a 463 control diet. Each bar represents the mean (± SEM) transcript abundance of IGF genes. Bars 464 denoted by different letters represent significantly different means (P < 0.05). 465 466 Figure 2: Hepatic transcript concentrations of genes of the IGF system in fetuses (B and C) 467 and sows (A) fed with a control or Fusarium contaminated diet on the 35 th to the 70 th and the 468 75 th to the 110 th day of gestation. Each bar represents the mean (± SEM) transcript 469 concentrations of IGF genes of each experimental and control group. Bars denoted by 470 different letters represent significantly different means (P < 0.05). 471 472

Figure 1 :Figure 2 :

 12 Figure 1: Normal hepatic transcript concentrations of genes of the IGF system in fetuses/full-term piglets at days 70 and 110 and sows at day 70 of gestation. The sows were fed with a control diet. Each bar represents the mean (± SEM) transcript abundance of IGF genes. Bars denoted by different letters represent significantly different means (P < 0.05). 139x86mm (600 x 600 DPI)

Table 1 .

 1 Composition of the experimental diets (g/kg)

		Food Additives and Contaminants
		Experiment 1	Experiment 2
		CON MYCO CON MYCO
	Components:
	Control triticale	150	0
	Contaminated triticale	0	150
	Control wheat	250	250	400	0
	Contaminated wheat			0	400
	Barley	284.3	284.3	284.3 284.3
	Dried chips	100	100	100	100
	Soybean meal	175	175	175	175
	Soy oil	10	10	10	10
	Vilomin 19730 a	30	30	30	30
	L-lysin HCL	0.7	0.7	0.7	0.7
	Calculated composition:
	Dry matter	875	875	884	877
	Crude protein	167	167	159	162
	Deoxynivalenol (mg/kg) b	0.15	4.42 0.21	9.57
	Zearalenone (µg/kg) b	3.5	48.3	4	358
	CON= control group; MYCO=Fusarium toxin contaminated group

a Premix provided per kg diet: Ca, 6.3 g; P, 2.1 g; lysine, 0.6 g; Na, 1.8 g; Mg, 0.3 g; vitamin A, 15000 IE; vitamin D 3 , 1500 IE; vitamin E, 45 mg; Cu, 22.5 mg; Se, 0.36 mg b Measured by HPLC-methods Page 16 of 19 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 2 .

 2 Primers used for cDNA synthesis and qPCR.

	Page 17 of 19		Food Additives and Contaminants
	Genes	Forward primers	Reverse primers	Primers for cDNA synthesis	size	Acc. Nos.
					(bp)
	Igf1	GCACATCACATCCTCTTCGCATC	AGCTCCGGAAGCAGCACTCATC	ATCCTGCCAGTGGCATGTCATTC 212 NM_214256
	Igf1r	GACTGGTCCCTGATCCTGGATG	CTCGGGGTGGCAGCACTCGTG	GCACTCGCCATCATGGATCAC	237 NM_214172
	Igf2	GCCTTGGCCTCGTGCTGCTATG	CAGCTACGGAAGCAGCACTCTTC	AGGTGTCATAGCGGAAGAACTTG 176 NM_213883
	Igf2r	GCCTCCAGATCTCTTCTGGAATTC GCTCTCTGTCAAACACGTAGCAC	AAGGGACAGGCACGCAACTGTG	232 AF339885
	Igfbp1 TCCTGGACAGTTTCCACCTGATG	ATAGAATCCGTTCTTGTTGCAGTTC CGTGGAGCCCAGGATCTTCTTC	236 AB053605
	Igfbp2 CCCGAGCAGGTTGCAGACAATG	GCGGCGGCCGCAGCTTCTTG	GTTCCAGGACCTGGTCCAACTC	235 NM_214003
	Igfbp3 GAGCACGGACACCCAGAACTTC	GAAGCCCTTCTTGTCGCAGTTAG	GAGGGGCTGCCCGTACTTATC	157 J05228

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgments 302

The authors are grateful for support through a grant from the Deutsche 303 Forschungsgemeinschaft (Ti 189/5-3; DA 558/3-3) and from the technical assistance of Mrs. 304 M. Anders, P. Reckling, and V. Tesch. 305


Lara La Pera

Giuseppe Avellone

Vincenzo Lo Turco

Giuseppa Di Bella

Pasquale Agozzino

Giacomo Dugo

La Pera Lara

Avellone Giuseppe

Turco Lo

Vincenzo

Di Bella Giuseppa

Agozzino Pasquale

Dugo Giacomo

Influence of roasting and different brewing processes on the ochratoxin A 1 content in coffee determined by HPLC-FLD. 2

Keywords: brewing methods, coffee, HPLC-FLD, ochratoxin A

A rapid and reliable procedure has been developed for the determination of ochratoxin A (OTA) in 11 green and roasted coffee. The method consists of extraction of the sample with methanol -5% 12 aqueous sodium hydrogen carbonate/1%PEG8000-(20:80), followed by IAC clean up and finally, 13 HPLC determination with fluorimetric detection. Mean recoveries for green and roasted coffee 14 spiked at different levels ranging from 94 and 105 % were obtained. The limit of determination 15 (S/N= 3) was 0.032 ng/g and the precision (within-laboratory relative standard deviation) was 6%.

16

The method described has been used to assess the influence of roasting and different brewing 17 processes on OTA content in commercial lots of green and roasted coffee. The results provided 18 evidence that roasting led to a significant drop on OTA levels (65-100%). Also the way coffee is 19 prepared affects the OTA content: brewing using a moka express (Italian coffee) led to a significant 20 reduction of OTA concentration (50-75%) since hot water stays in contact with coffee for a short 21 time, on the contrary Turkish coffee making (infusion for about 10 min) cause poor reduction in 22 OTA 23

The occurrence of ochratoxin A (OTA) in raw coffee has been reported in various studies since 27 1974 [START_REF] Levi | Study of the occurrence of ochratoxin A in green coffee 281 beans[END_REF]Micco et al, 1999;[START_REF] Nakajima | Survey of aflatoxin B1 and ochratoxin A in 287 commercial green coffee beans by high performance liquid chromatography linked with 288 immunoafinity chromatography[END_REF][START_REF] Tsubouchi | A survey of occurrence of mycotoxins and 310 toxigenic fungi in imported green coffee beans[END_REF]Truckness et 28 al, 1999). OTA is a mycotoxin produced as a metabolic product of certain fungi, mainly of the 29 genera Aspergillus and Penicillium. OTA toxicity is mainly a result of interactions with nucleic 30 acids, determining deleterious effects on the protein synthesis and integrity of nucleic acids with 31 consequent damage to the organisms; for these reasons, OTA is considered one of the most 32 powerful natural hepatocarcinogenic agents [START_REF] Chu | Mycotoxins: Food contamination, mechanism, carcinogenic potential and preventive 266 measures[END_REF][START_REF] Galvano | Mycotoxins in the human food chain[END_REF]. The 33 carcinogenicity of OTA has been evaluated by the IARC in a recent monograph where OTA is 34 classified as Group 2B, possible human carcinogen (IARC, 1993) 35 36

The EU has fixed legal limits for the occurrence of Ochratoxin A (OTA) in following foods: cereals 37 (5 ng g -1 ), products derived from cereals (3 ng g -1 ), wine and grape juice (both 2 ng g -1 ) dried vine 38 fruit (currants, raisins and sultanas all 10 ng g -1 ), and from 2005 also roasted coffee (5 ng g -1 ) and 39 soluble coffee (10 ng g -1 ) (EU Regulation 123/2005). The growth of mould on coffee beans is 40 possible if the moisture content of the beans exceeds the accepted standard for an extended time 41 (Studer-Rohr et al, 1995). An Italian study carried out by Romani et al in 2000 provided evidence 42 that 106 out of 162 green coffee samples coming from different countries (84 from Africa, 60 from 43 America, and 18 from Asia) were contaminated by OTA, with concentration ranging from 0 to 48 44 ppb. In particular, it was possible to verify that, samples from African countries were more 45 contaminated as compared to samples from other origins in terms of frequency and level of OTA. Over the past few years, the sensitivity of analytical techniques for the detection of OTA has 75 improved. The most diffused technique for OTA analysis in food is liquid chromatography with 76 fluorescence detection (LC-FLD), preceded by sample purification by using immunoaffinity 77 columns [START_REF] Pittet | Liquid Chromatographic Determination of 293 Ochratoxin A in pure and Adulterated Soluble Coffee Using an Immunoaffinity Column Cleanup 294 Procedure[END_REF][START_REF] Sibanda | Optimization of solid-phase clean-up prior to 301 liquid chromatographic analysis of ochratoxin A in roasted coffee[END_REF], but also the use of different types of columns was 78 described [START_REF] Ventura | Analysis of 320 Ochratoxin A in Coffee by Solid-Phase Cleanup and Narrow-Bore Liquid Chromatography-321 Fluorescence Detector-Mass Spectrometry[END_REF]. In this study OTA extraction from green and roasted coffee was 79 carried out using methanol-5% aqueous sodium hydrogen carbonate /1% PEG8000 (20:80) 80 followed by immunoaffinity clean up; this procedure was compared with the one described by Pittet 81 et al (1996) which is the most reliable and diffused analytical method to measure OTA levels in 82 green and roasted coffee.

HPLC-FLD conditions

98

The separation of OTA was achieved with a Phenomenex Luna C18 Column (150x4.6 mm, 5µm 99 particle size) and a guard column packed with the same material using a mobile phase composed of

Extraction with methanol -5% aqueous sodium hydrogen carbonate/1%PEG8000-(20:80)(A)

115 10 g of grounded roasted or green coffee were mixed with 100 mL of methanol-5%NaHCO 3 / 1% 116 PEG8000 (solution A) and placed in a ultrasound bath at 50°C for 10 min. After this time the 117 mixture was filtered trough filter paper, then 10 mL of the filtrate were transferred in a graduate 118 cylinder and diluted to 20 mL with a solution of 5%NaHCO 3 -1 % PEG8000 and loaded onto the 119 IAC.

120

Extraction with methanol-3% aqueous sodium hydrogen carbonate (50:50)(B)

121 This extraction method was similar to the one described by [START_REF] Pittet | Liquid Chromatographic Determination of 293 Ochratoxin A in pure and Adulterated Soluble Coffee Using an Immunoaffinity Column Cleanup 294 Procedure[END_REF]. 10 g of grounded 122 roasted coffee were mixed with 100 mL of methanol-3% aqueous sodium hydrogen carbonate The results from recovery test ( blank samples with OTA covering the range from 0.05 to 50 ng/g (Fig. 3). The limit of detection 185 (LOD) and the limit of quantification (LOQ), calculated as 3-and 10-fold the signal to noise ratio, 186 were 0.032 and 0.107 ng/g, respectively, lower than those found by [START_REF] Pittet | Liquid Chromatographic Determination of 293 Ochratoxin A in pure and Adulterated Soluble Coffee Using an Immunoaffinity Column Cleanup 294 Procedure[END_REF], van der 187 [START_REF] Stegen | Effect of roasting conditions on reduction of 303 ochratoxin A in coffee[END_REF], [START_REF] Sibanda | Optimization of solid-phase clean-up prior to 301 liquid chromatographic analysis of ochratoxin A in roasted coffee[END_REF]. The method described fits with the requirements of diastereomer, and thermal degradation [START_REF] Boudra | Thermostability of Ochratoxin A in Wheat under Two 261 Moisture Conditions[END_REF]Studer-Rohr et al, 1995). In this case the 231 results obtained revealed first-order reaction (y=-0.024x, R 2 = 0.918), whereas a more complex

  , water, methanol were purchased from Romil (Cambridge, GB). The OTA 87 standard was purchased from Sigma (Sigma-Aldrich, Milan, Italy). Samples were filtered trough a 88 Sartorius PES 0.2 µm membrane (Goettingen, Germany). A standard solution of OTA was prepared 89 from the OTA vial purchased from Sigma. The standard stock solution was made in 4 mL of 90 methanol at 250 µg/mL, and stored at -20 °C. An intermediate standard solution was prepared at 10 91 µg/mL, by diluting 1 mL of stock standard solution with 25 mL of methanol. All of the solutions 92 were stored at -20 •C. The RIDA IACs were obtained from Bio-Pharm (Darmstadt, Germany). 93 Apparatus 94 An Agilant 1100 Series liquid chromatograph equipped with a G1312A binary pump, a G1321A 95 fluorimetric detector, and a Rheodyne 7125 injection valve fitted with a 20 µl loop, a Phenomenex 96 (Terrance, CS, USA) Thermasphere column oven, was used.

  97

  123(50:50) (solution B) and placed in a ultrasound bath for 10 minutes. After this time the mixture was 124 filtered trough filter paper, then 10 mL of the filtrate was transferred in a graduate cylinder and 125 diluted to 50 mL with PBS. Aliquots of 20 mL were passed over the IAC. allow solvent evaporation prior to extraction. To study the between-day precision of the 153 method, OTA fortified (2 ng/g) samples, were analysed in triplicate for 3 days. increasing demand for new OTA extraction techniques from coffee, cereals, and many 158 other food matrices, with shortened extraction times and reduced costs which ensures high 159 recovery, precision and low limit of detection (LOD) of the toxin. In 2007 Liazid et al. described 160 the stability of OTA during its extraction using different extraction techniques, including 161 ultrasound-assisted extraction (UAE) and magnetic stirring assisted extraction (MSAE), applied to a 162 model solution. In this study we tested the use of ultrasound for OTA extraction from coffee using 163 two different solvents: methanol -5% aqueous sodium hydrogen carbonate /1% PEG8000 (20:80) 164 (solution A) and methanol-3% aqueous sodium hydrogen carbonate (50:50) (solution B). The effect 165 of ultrasound is to help penetration of the solvent in the sample matrix and to promote the 166 dissolution of OTA in the extracting solvent (Liazid et al, 2007). Figure 1 provides evidences that 167 the application of ultrasound at 50 C ° for 10 min. ensures good recoveries both using solution A 168 (99.0±0.70%) or solution B (95.0±0.73%) as the extraction solvent from an artificially 169 contaminated coffee spiked with 2 ng/g of OTA. No significant influence of temperature (40, 50 170 and 60 °C ) was found (data not shown). Therefore further experiments were performed by 171 extracting OTA with solution A in a ultrasound bath for 10 min. at 50C°. The extraction procedure 172 with solution A plus IAC clean-up provided a clean LC-FLD chromatogram for green and roasted 173 coffee (flat baseline, practical lack of impurity peaks) due to IAC selectivity, as can be seen in Fig.

  215 also be explained by the short duration of the process. An average OTA reduction of 20 % was 216 found after infusion of grounded coffee in hot water for 10 min (Turkish coffee); in this case the 217 time spent in contact with water is longer therefore higher amounts of the toxin were about the behaviour of OTA in hot water and to assess a possible interaction of 221 the toxin with the substrate during coffee brewing, the following experiments were undertaken: a) 222 kinetics of OTA in pure hot water (water temperature was kept at 95-96°C over time) b) reduction 223 of OTA by brewing coffee in hot water over time (the temperature was kept at 95-96C° over time) 224 using a naturally contaminated sample with 2.1 ng g -1 c) reduction of OTA by brewing coffee in 225 hot water (water at 95 C° was poured onto grounded coffee an it was let to cool during the 226 experiment; after 45 min. the temperature was 40 °C).Figure 4 shows that OTA content in pure 227 water (a) after 2 and 5 min. was reduced by 5-15%; after 10 min OTA decrease was of 23%, 228 whereas by prolonging heating up to 45 min OTA levels decreased by 44-61%. Two different 229 explanations are available for this reduction: isomerization at the C-3 position into another 230

Figure 1 Fig. 2 .

 12 Figure 1 Effect of ultrasound application time at 50 °C on OTA extraction from an artificially contaminated coffee spiked with 2 ng/g of the toxin. Each point represents a mean of three replicates, the RSD% of the measures was of 0.75%. A: extraction with methanol -5% aqueous sodium hydrogen carbonate/1%PEG8000-(20:80) B: extraction with methanol -3% aqueous sodium hydrogen carbonate (50:50)

Figure 3 Figure 4

 34 Figure 3 Calibration curve. Each point is the mean of three replicates, the RSD% of the measures is

  Roasters operate at temperatures of 200°C for 3 minutes. Experiments were carried out 110 using 14 lots of 650 kg of green coffee beans coming from different countries (4 from India, 5 from

	101	CH 3 CN/H 2 O/CH 3 COOH (54:44:2, v/v) at a flow rate of 1mL/min; the column temperature was
	102	30C°. Fluorescence excitation and emission wavelengths for OTA detection were 334 and 460 nm,
	103	respectively; an adequate signal-to-noise ratio was obtained with a photomultiplier gain of 14 and a
	104	response time of 4 sec.
	105	Samples
	106	Samples were cleaned, roasted, cooled, ground, and packaged in a Sicilian firm (ZiCaffè, Marsala,
	107 108	F Italy). Bags of green coffee beans are machine-opened, dumped into a hopper, and screened to o r remove debris. The green beans are then transferred by belt to storage hoppers. From the storage
	109	hoppers, the green beans are conveyed to the roaster. 240 Kg of green coffee were simultaneously P e roasted.
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111

Vietnam, 2 from Cameroun, 1 from Ethiopia, 1 from Brazil and 1 from Salvador). Nine 112 commercial soluble coffee samples bought in a supermarket in Palermo (Italy) on July 2007 were 113 also analysed.

114

Table 1 )

 1 provided evidence that the extraction of OTA with solution et al. (2001). The higher recoveries obtained in this study may be explained by the use of PEG 8000 179 and higher NaHCO 3 percentage in the extraction and diluting solutions as reported by Visconti et al 180 (1999) in a study concerning OTA extraction from wine. The between-day coefficient of variation,

	181
	182	which is very important when establishing routine methods to be used over extended periods, for
	183	method A was of 3.6%; precision calculated as RSD% of nine measurements of a spiked sample (2
	184	ng/g), was 6%. A calibration curve (y=3.2997x, R=0.9984) was obtained spiking homogenised
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176

A yielded higher levels of the toxin (by 1-8%) compared to those obtained using methanol-3%

Table 1

 1 Mean recoveries obtained for OTA extraction with methanol-5% aqueous sodium hydrogen carbonate/1%PEG8000 (20:80) (A) and extraction with methanol-3% aqueous sodium hydrogen carbonate (50:50) (B) from blank and naturally contaminated samples

			Food Additives and Contaminants	Page 18 of 20
	SAMPLE	OTA (ng/g)	OTA added	Recovery %	Recovery %
			(ng/g)	(A)	(B)
		nd	0.5	94.0	93.1
		nd	2.0	95.0	93.0
		nd	4.0	96.5	95.5
	ROASTED	nd	10.0	98.4	91.0
		0.930	2.0	95.0	95.0
		0.930	4.0	99.5	95.1
		0.930	10.0	102.0	94.3
		nd	2.0	105.1	98.0
		nd	5.0	99.0	95.1
	GREEN	nd	15.0	96.0	92.4
		2.2	2.0	98.0	92.0
		2.2	5.0	98.1	94.0
		2.2	15.0	95.2	94.3

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Table 2

 2 Range levels of OTA in green and roasted coffee samples from different origins *Reduction was significant at t <0.0001

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Food Additives and Contaminants

aqueous sodium hydrogen carbonate as suggested respectively by[START_REF] Pittet | Liquid Chromatographic Determination of 293 Ochratoxin A in pure and Adulterated Soluble Coffee Using an Immunoaffinity Column Cleanup 294 Procedure[END_REF] and van der Stegen

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different extraction techniques Talanta 71: 976-980

Acknowledgements


Torben ; Leth

Sisse Fagt

Steffen Nielsen

Keywords: Exposure assessment, Health significance, Risk assessment, Survey Additives/Contaminants: Nitrate, Nitrite Food Types: Meat, Processed foods Estimated intake, sodium nitrite, sodium nitrate, Denmark, cured meat, children

Nitrite and nitrate content in meat products and estimated intake in Denmark from 1998 to 2006

 I. Food groups in the Danish Dietary Survey and most abundant foods analysed for nitrite.

Table II. Sodium nitrite mgkg -1 . Mean for samples with content > detection limit (n=number of samples), (min-max).

Table III. Sodium nitrate mgkg -1 . Mean content in all samples and mean for samples with content > detection limit (n=number of samples), (min-max).

Table IV. Mean intake of sodium nitrate from cured meat mgday -1 based on samples with content of nitrate (high intake calculation). Table V. Food intake and high intake of NaNO 2 in 2006. Age group 4-5 years.

Abstract

The content of nitrite and nitrate in cured meat products has been monitored in Denmark seven times in the period from 1995 to 2006. The maximum permitted added amounts of sodium nitrite in Denmark (60 mgkg -1 for most products up to 150 mgkg -1 for special products) was not exceeded, except for a few samples in 2002. The intake, mean and intake distribution, of sodium nitrite was calculated from 1998 to 2006 with data from the Danish dietary survey conducted in 2000-2002 on Danes from 4 to 75 years of age. The amounts used by industry were relatively stable through the whole period with levels varying between 6 and 20 mg sodium nitrite kg -1 with sausages, meat used for open sandwiches and salami type of sausages as the greatest contributors. The mean intake of sodium nitrate was around 1 mg day -1 , which is very low compared to the total intake of 61 mg day -1 . The mean intake of sodium nitrite was in mg kg -1 bw day -1 for men and women was 0.017 and 0.014, 0.009 and 0.008 and 0.007 and 0.003 in the age groups 4-5 years, 6-14 years and 15-75 years, respectively, which was much lower than the ADI value of 0.09 mg kg -1 bw day -1 . The 99 percentile for the group of 4 year olds was 0.107 and 0.123 mg kg -1 bw day -1 for boys and girls, respectively, and the 95 percentile 0.057 and 0.073 mg kg -1 bw day -1 for boys and girls, respectively, highest for the girls. With less than 100 boys and girls in the age group 4-5 years only a small number of individuals were responsible for the high intake. The conversion of nitrate to nitrite in the saliva and the degradation of nitrite during production and storage must also be considered when evaluating the intake of nitrite.

Introduction

Nitrite, used as a food additive for its antimicrobial properties in cured meat products and its desirable effects on colour, flavour and texture, is considered a considerable health risk with high exposure caused by its ability to form carcinogenic nitrosoamines in foods and in the organism (SCF 1995). The ADI value is fixed at 0.06 mg nitrite (nitrite ion) or 0.09 mg sodium nitrite kg -1 bw day -1 . For nitrate the ADI value is fixed at 3.7 mg nitrate (nitrate ion) or 5 mg sodium nitrate kg -1 bw day -1 .

EU controls as specified in Directive 95/2/EC (EU 1995) are based on fixed residual amounts between 50 and 175 mg kg -1 of sodium nitrite (in bacon) and an indicative usage amount of 150 mg kg -1 . In Denmark stricter rules have been employed based on permissible added amounts from 60 mg to 150 mg kg -1 of sodium nitrite (the higher amount only for semi-preserved products and special cured hams) [START_REF] Positivlisten | Fortegnelse over tilsaetningsstoffer til fødevarer[END_REF], and we succeeded in continuing with these rules after winning a case against the Commission at the European Court of Justice in 2003 (Opinion, EFSA 2003). In Denmark nitrate is only allowed added to bacon of the Wiltshire type and special types of ham in an amount of 300 mg kg -1 , while in 95/2/EC a residual amount of 250 mg kg -1 in salted meat products is specified.

In Estonia an average estimated intake in 2003-04 of 105% of the ADI value of 0.06 mg nitrite kg -1 bw day -1 for the 1 to 6 year age group was found [START_REF] Reinik | Nitrites, nitrates and N-nitrosoamines in Estonian cured meat products: Intake by Estonian children and adolescents[END_REF]. In New Zealand the average intake has just recently been estimated to 13% of the ADI with maximum intake 7 times this value, and the authors concluded that with the endogenous conversion of nitrate to nitrite, between 10% and 50% of all people will exceed the ADI (Thomson et al. 2007), both investigations indicating a high intake of nitrite with many children having intakes over the ADI value. In Denmark as a part of the Danish Veterinary and Food Administration programme for monitoring of food additives the content of nitrate and nitrite in meat products has been investigated seven times between 1995 and 2006.

The primary objective has been to check that the additives are used legally and within the maximum permitted levels, but the analyses are also utilized to assess the intake of food additives. A rough average estimate of the intake of nitrite and nitrate has been carried out earlier giving a value of 0.5 mg nitrite day -1 , which even for small children is well below the ADI value, and 61 mg nitrate day -1 . Cured meat was the most important source for nitrite with an additional 30% contribution from fruit and vegetables, while for nitrate fruit and vegetables and water were the only important sources with only 1 mg day -1 coming from meat (Petersen and Stolze 1999). However, an intake distribution could not be calculated.

With the new dietary survey carried out by the National Food Institute from 2000 and onwards [START_REF] Lyhne | Dietary habits in Denmark 2000-2002[END_REF] and the data from the monitoring programme it is possible to estimate the intake and intake distribution. The results of the development of the use of nitrite and nitrate in cured meat in Denmark and the intake estimation for nitrite and nitrate are presented in this paper.

Materials and methods

Monitoring programme

The Danish food monitoring programme for food additives started in 1995 with the purpose of partly supervising the actual occurrence and use of food additives, partly controlling whether the maximum permitted levels were exceeded and whether illegal applications and/or declaration errors occurred in contravention of EU rules. Since the beginning meat and meat products have been analysed in seven different years from 1995 to 2006.

Samples have been drawn all over the country from producers, importers and wholesalers with production both Danish produced and imported. The most abundant meat products in the food groups from the dietary survey are shown in table I and the total number of samples are shown in table II. The samples were sent to the laboratory and stored in a refrigerator at 4ºC. If analysis could not be performed within two days after receipt, the samples in unopened condition were frozen in polyethylene bags at -18ºC until analysis could be started.

Chemical analysis

Nitrite and nitrate were extracted from the samples by mixing 5 min with hot water (70ºC).

Protein was precipitated with addition of Carrez solution I and II and the suspension was filtrated. The filtrate was injected in a FIA system, where sulphanilamide and N-(1-naphtyl)ethylenediammoniumchloride were added to the carrier stream to react with nitrite to form a violet azo colour which was measured spectrophotometrically at 540 nm. For nitrate, after injection, the carrier stream went through a Cd-column where nitrate was reduced to nitrite which subsequently reacted to form the azo colour. A calibration curve was prepared every day with nitrite and nitrate on 5 concentration levels for quantitative calculation. All chemicals were analytical grade and the water was de-ionised. The laboratory was accredited to perform the analysis after ISO 17025. In every series of samples was included blind samples of reagents and water, a duplicate divided between two series, a reference material of freeze dried dill and a recovery experiment for both nitrate and nitrite with the data plotted in control charts. Depending on sample type and concentration level an average recovery of 100% for nitrate and 95% for nitrite was found and relative standard deviations between 2 and 4% for nitrite and between 2 and 5% for nitrate.

Detection limit for NaNO 2 was 3 mgkg -1 and for NaNO 3 5 mgkg -1 . The results showed that the analytical method had been under control at all times with no values exceeding the control limits for double determinations or for recoveries.

Dietary survey

The survey was conducted in 2000-2002 on a random sample of Danes aged from 4 to 75 years drawn from the Central Population Register [START_REF] Lyhne | Dietary habits in Denmark 2000-2002[END_REF]. Dietary data was based on recording on 7 consecutive days. The questionnaire was designed to cover the whole diet and included 5 cuts of meat used on bread (e.g. different types of meat, salami and liver paste), 3 types of pork meat (lean, medium and high fat) and sausages. A combination of food quantification methods (household measures and estimation from photographs of different portion sizes) were used to obtain the best estimate of amounts consumed.

Estimated intake calculation

The between 160 and 320 samples of meat analysed in the later years were aggregated into eight different types of meat for which intake data was present: Liver paste; paté; fatty meat intended to be used in sandwiches; lean meat intended to be used for sandwiches; salami; sausages for dinner; medium fat and fat pork meat for dinner.

For each person the registered average daily intake of the eight food groups or the part of the food group with content of nitrite (see table I) was multiplied with the content of nitrite, and a calculation of basic statistic parameters of the intake distribution was carried out in the form of mean, standard error of mean and percentiles with the persons divided in age groups 4-5 year, 6-14 year, 15-75 year, males, females and the total population.

For the content of nitrite two scenarios were used: One with the analytical determined mean value used for all the samples with a detectable nitrite content called "high intake", and another with the analytical determined values for all the samples in each category called "average intake", which will be lower than "high intake", since many of the meat samples were produced without the use of nitrite salts. The variability in the content of nitrite within each meat category was not used in the estimated intake calculation.

Results

[Insert Table I about here]

In table I is shown the eight food groups where data were collected in the dietary survey and the most abundant foods analysed for nitrite. It was estimated based on knowledge of dietary habits in Denmark and sales statistics that 5% of liver paste was eaten as paté, that 7-10% of fat pork meat was eaten as bacon, which was also used in fat meat for open [Insert Table II about here] In table II is shown the mean sodium nitrite content found in samples with content over the detection limit. The mean value of all the samples is typically about 20% lower than the mean value of samples with content over the detection limit (data not shown). In very few samples the maximum permitted level of nitrite was exceeded and that was back in 2002 and earlier. A few declaration errors were found every year mainly due to declaration of nitrite, which could not be found in the sample, probably because the nitrite was used up during production and storage. No nitrite was used in liver paste, where it is not allowed. However, in paté nitrite was generally used. In the group meat for open sandwiches, fat and medium fat nitrite was found in more than half of the samples. However, the results show that it was also possible to produce meat products in these groups without using nitrite. Products like bacon and sausage of the saveloy type often contained nitrite, while smoked saddle and loin fillet were often without added nitrite. Salami was mainly without nitrite, while the other sausage types in this group often contained nitrite. Also sausages usually contained nitrite. The average level of nitrite seemed fairly stable over the years although with some variation and with a low level in 2005. However, in 2006 the level of nitrite increased again.

[Insert Table III In table III is shown the average nitrate content in samples with content over the detection limit but only for the years 2005 and 2006. The nitrate content was generally low but higher than a possible natural content, which meant that added nitrate was found in many meat products, where it was not allowed to add nitrate. However, in many cases it could not be excluded that the nitrate followed an ingredient, where addition was legal.

[Insert Table IV about here]

In table IV is shown the average sodium nitrate intake in the age groups 4-5 year, 6-14 year and 15-75 year with the contribution from the different food groups for the years 2005 and 2006 based on the average of the samples with nitrate content, that is, a high intake calculation. It appears that the average intake of nitrate from meat was more or less the same in all age groups and both years about 1 mg day -1 , although a little higher in 2006 than 2005, which was very low compared to the intake from vegetables, fruits and water.

Fifty percent of the samples did not comply with regulations either through mislabelling or addition of nitrate, where it is not permitted. No improvement has been seen with this situation, which has persisted for a number of years despite follow-up with producers and importers. This necessitates a continuation of the monitoring programme for nitrite and nitrate in meat products. A detailed intake distribution of sodium nitrite in 2006 is shown in table V for the age group 4-5 year. The mean for samples with content over the detection limit (high intake calculation) was taken from table II. For bacon in the food group fat pork meat was used the content for fat meat for open sandwiches, and for smoked pork saddle and ham in the food group medium fat pork meat was used the content for lean meat for open sandwiches. An overview of the calculations for all the years from 1998 to 2006 and all the age groups appears in figure 1.

[Insert Table V]

[Insert figure 1 about here] Only for 4-5 year olds the intake was rather high and even with the 99 percentile (P99) exceeding the ADI value. However, with the number of participants below 100 it is a single individual, who determined P99, and apparently there was always at least one person with unusual food consumption. 4-5 year old girls had higher P99 and P95 than boys because of a higher intake of sausages, but in the other age groups men had a higher intake of nitrite than women because of a higher food intake.

[Insert figure 2 and 3 about here] From figure 2 and 3 the significant sources for nitrite intake are demonstrated. Sausages, fat and lean meat for open sandwiches (the famous Danish "smorrebrod") and salami type of sausage were the main sources in that order. The 4-5 year old girls had a higher consumption of sausage than the 4-5 year old boys. On the other hand boys eat more fat meat for open sandwiches than the girls. The distribution was more or less the same for all percentiles as for the mean intake.

Discussion

Data for nitrite intake in table V and figure 1 are only shown for "the high intake" calculation based on the consideration that people do not eat an average diet, but they have probably considerable brand loyalty and will eat the same sausage or ham as they always eat. This may be with or without nitrite, but "the high intake" calculation will be a more realistic representation of what people can be exposed to.

The intake of nitrate of about 1 mg per day in 2005 and 2006 (see table IV) was very low compared to the intake from vegetables, fruit and water with an average intake in Denmark of about 61 mg nitrate day -1 [START_REF] Petersen | Nitrate and nitrite in vegetables on the Danish market: content and intake[END_REF]. It can be concluded that the intake of cured meat is not important for the nitrate intake, and therefore intake so the decision seems not to have influenced the use of nitrite in Danish cured meat products. This is not a surprise, since the maximum permitted level did not change.

The total intake of sodium nitrite with cured meat varied from a mean value around 0.3 mg day -1 to P99 of 2-3 mg day -1 , and no clear connection between age and intake can be seen, except that 15-75 year old men seem to have a higher total intake than the other age groups (see table V and figure 1). This means that the young 4-5 year old children had a P99 for nitrite intake 20 to 35% over the ADI value and also P95 was high for both boys and girls. However, these values were based on very few children with less than a hundred boys and girls in the 4-5 year groups.

Compared to Reinik [START_REF] Reinik | Nitrites, nitrates and N-nitrosoamines in Estonian cured meat products: Intake by Estonian children and adolescents[END_REF], where the mean intake in Estonia for 1-6 year old children in 2004 was about 50% of the ADI value based on all children and all days, the intake in Denmark is much lower with mean intake 16-19% of the ADI value. In Estonia the maximum permitted value for residual sodium nitrite was 100 mgkg -1 in contrast to the Danish system with maxium permitted added levels, mostly only 60 mgkg -1 . In New Zealand (Thomson et al. 2007) a mean sodium nitrite intake for adults of 13% of the ADI value was recently found based on the content in both meat and vegetables compared to a mean intake in Denmark of only 5% of the ADI value for adults. Only in broccoli was found a nitrite content over the detection limit, but for other vegetables the intake was calculated with a level of half the detection limit, i.e. 2.5 mgkg -1 . In the Danish investigation from the nineties [START_REF] Petersen | Nitrate and nitrite in vegetables on the Danish market: content and intake[END_REF] an intake of 0.097 mgday -1 sodium nitrite from vegetables was found, which amounts to about 30% of the intake found with cured meat in this investigation, but no attempt has been made here to correct the intake with these old data from 1996 for nitrite content in vegetables.

Another aspect to consider is the fact that there is no direct relationship between the ingoing and the residual amount of nitrite, but the degradation of nitrite added to meat is influenced by several factors like pH-value, storage temperature, heat treatment of the meat and the presence of reducing substances. Especially added ascorbate will increase the rate of degradation of nitrite. (Opinion, EFSA 2003). Thus, ideally addition of nitrite should be controlled through recipe control of maximum permitted added levels, instead of analytical control of maximum permitted added levels, as it has been done in this investigation, or maximum residual amounts after directive No 95/2/EC, as it has been done in many other investigations.

Endogenous conversion of nitrate in saliva to nitrite is reported to be between 5% and 20% of the nitrate (SCF 1995). With a mean intake of 60 mg nitrate day -1 this amounts to 3 mg nitrite day -1 , which is higher than even the P99 intake value for nitrite with cured meat. The 4-5 year old will get 0.15 mgkg -1 bwday -1 sodium nitrite, which is about 50% over the ADI value of 0.09 mg, and the 6-14 year old will get 0.08 mgkg -1 bwday -1 , which together with the intake from cured meat also will exceed the ADI value for more than 10% of the group.

Only for the adults, who will get 0.04 mgkg -1 bwday -1 , will the conversion of nitrate to nitrite not lead to transgression of the ADI value. If the calculation is carried out with the P95 for nitrate intake of 113 mgday -1 [START_REF] Petersen | Nitrate and nitrite in vegetables on the Danish market: content and intake[END_REF] and/or a conversion of 20% of nitrate to nitrite, much higher intakes of nitrite will be obtained. However, it is known that higher contents of nitrite will also increase the content of nitrosamines in meat products, but whether such a relation exists for endogenous formation of nitrosamines from precursors is presently not known. So far no epidemiological studies have shown a correlation between increased nitrate or nitrite intake and increased cancer risk and may even show positive health effects due to the positive effects of high intake of fruit and vegetables (SCF 1995).

With these uncertainty factors taken into account, which point to an underestimation of the nitrite intake based on residual amounts, the advice remains to keep the level of nitrate and nitrite in food as low as the minimal required to achieve the necessary preservative effect.

Conclusion

Monitoring the added amounts of nitrate and nitrite to cured meat from 1998 to 2006 showed a relatively stable use of nitrite as a food additive with a slight decrease in 2005 but increase again in 2006. The levels varied in the different food groups from 6 to 20 mg kg -1 of sodium nitrite for sausages, fat and lean meat for open sandwiches, and salami type of sausages with the highest content. With the Danish Dietary Survey from 2000 to 2002 the mean intake and intake distribution have been calculated in three age groups 4-5 year, 6-14 year and 15-75 year for both men and women. This gave mean intakes well below the ADI value for sodium nitrite of 0.09 mg kg -1 bw day -1 . However, P99 for the group of 4-5 year olds exceeded the ADI value with about 30% and P95 was more than 60% of the ADI value, but with groups of boys and girls below a hundred very few persons with a special eating pattern were responsible for the high intakes. With a conversion of nitrate in the saliva to nitrite of at least 5% of the intake of nitrate, which in Denmark is about 60 mg day -1 , the nitrite from conversion will by far exceed the intake of nitrite from cured meat and also in many cases exceed the ADI value. The significance of conversion of nitrate to nitrite in relation to the existing ADI for nitrite of 0.06 mg kg -1 bw day -1 needs evaluation.

Figure 1 .

 1 Figure 1. Intake of NaNO 2 mgkg -1 bwday -1 .

Figure 2 .

 2 Figure 2. Distribution of mean nitrite intake. Boys 4-5 years.

Figure 3 .

 3 Figure 3. Distribution of mean nitrite intake. Girls 4-5 years.

  that 30% of medium fat pork meat was eaten as smoked pork saddle and ham, which were also used in lean meat for open sandwiches.

  calculations presented in this article have only been carried out in detail for 2005 and 2006. Considering the development in the content of nitrite from 1998 to 2006, with too few samples to include 1995 and 1997 in the evaluation, there was apparently a decreasing tendency towards 2005. However, in 2006 a small increase in the nitrite content was seen again. Thus, it seems the variation was more or less accidental. The Danish authorities have been very interested in the development in the use of nitrite after the decision in 2003 about the more strict Danish rules for the use of nitrite could be uphold (Opinion, EFSA 2003). However, no clear development can be seen after 2003 compared to before 2003,

  Report of the Scientific Committee for Food. Opinion of the Scientific Committee for Food on: Nitrates and nitrite. European Commission. Thomson BM, Nokes CJ, Cressey PJ. 2007. Intake and risk assessment of nitrate and nitrite from New Zealand foods and drinking water. Food Additives and Contaminants 24

Boys, n=82, Average weight 21.3 kg Food intake gday -1 High intake of NaNO 2 mgday -1

  Liver paste Liver paste and paté (5% of the food group) 2. Meat for open sandwiches, fat Pork loin, spiced pork flank, meat balls, pork sausage saveloy type, bacon 3. Meat for open sandwiches, lean Ham, roast beef, smoked pork saddle, salted veal eye of round,

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	Food group	4-5 year old	6-14 year old	15-75 year old
	Food group 1. Liver paste Food group 1. 5% paté 2. Meat for open sandwiches, fat Foods analysed for nitrite Mean P50 7.2 5.2 NaNO 3 intake mgday -1 NaNO 3 intake P90 P95 15.0 19.1 mgday -1 Food Food Food intake intake intake 59.4 P99 NaNO 3 intake Mean P50 0 0 mgday -1 0.4 0.3 0.8 1.0 3.0 0.002 0.002 0.005 0.006 P90 P95 0 0 5.1 2.1 12.9 15.6 24.6 0.095 0.040 0.243 0.294 3. Meat for open sandwiches, lean 3.5 1.5 10.3 12.9 27.9 0.055 0.022 0.158 0.198 4. Salami 4.1 3.3 11.6 12.9 17.9 0.045 0.036 0.126 0.140 5. Sausages 11.2 5.0 30.0 40.0 78.6 0.130 0.058 0.348 0.464 6. Pork meat, fat 3.1 0 11.6 15.7 43.3 0 0 0 0 6. 7% bacon 0.2 0 0.8 1.1 3.0 0.004 0 0.015 0.021 7. Pork meat, middle fat 3.4 0 11.6 15.7 34.6 0 0 0 0 7. 30% smoked pork saddle and ham 1.0 0 3.5 4.7 10.4 0.016 0 0.053 0.073 8. Pork meat, lean 1.1 0 4.3 11.6 14.4 0 0 0 0 Total intake of NaNO 2 mgday -1 0.347 0.158 0.949 1.196 Intake of NaNO 2 mgkg -1 bwday -1 0.016 0.007 0.045 0.056 Girls, n=77, Average weight 20.3 kg 1. Liver paste 7.0 6.4 15.0 22.9 46.7 0 0 0 0 1. 5% paté 0.4 0.3 0.8 1.1 2.3 0.002 0.002 0.005 0.008 2. Meat for open sandwiches, fat 3,1 1.1 7.7 12.9 28.0 0.058 0.020 0.146 0.243 3. Meat for open sandwiches, lean 3.4 1.3 9.4 12.9 14.9 0.053 0.020 0.145 0.198 4. Salami 4.2 2.6 10.9 15.0 28.0 0.046 0.028 0.118 0.164 5. Sausages 10.6 4.3 37.1 48.6 117.1 0.123 0.050 0.431 0.563 6. Pork meat, fat 1.0 0 0 11.4 23.1 0 0 0 0 6. 7% bacon 0.1 0 0 0.8 1.6 0.001 0 0 0.015 7. Pork meat, middle fat 2.8 0 11.6 15.7 23.1 0 0 0 0 7. 30% smoked pork saddle and ham 0.8 0 3.5 4.7 6.9 0.013 0 0.053 0.073 8. Pork meat, lean 0.2 0 0 0 7.1 0 0 0 0 Intake of NaNO 2 mgkg -1 bwday -1 0.015 0.006 0.044 0.062 v i e e i v Total intake of NaNO 2 mgday -1 0.295 0.119 0.899 1.263 F o r P e e r R e gday -1 2005 2006 gday -1 2005 2006 gday -1 2005 2006 1. smoked pork loin fillet 4. Salami Salami, liver sausage, tea sausage, hunters sausage 5. Sausages Frankfurter, Danish pork sausage, cocktail sausage 6. Pork meat, fat Only bacon (7% or 10% of the food group) 7. Pork meat, medium fat Only smoked pork saddle and ham (30% of the food group) 8. Pork meat, lean Not analysed F o r P e e r R e O n l y 1. Liver paste 6.73 0.091 0.168 5.26 0.072 0.132 5.12 0.070 0.128 2. Paté 0.35 0.012 0.012 0.28 0.009 0.010 0.27 0.009 0.010 3. Meat for open sandwiches, fat 4.1 0.099 0.159 3.37 0.081 0.131 4.71 0.114 0.183 4. Meat for open sandwiches, lean 3.48 0.075 0.096 4.87 0.105 0.134 6.2 0.133 0.171 5. Salami 4.16 0.166 0.212 3.17 0.126 0.161 2.6 0.104 0.132 6. Sausages 10.91 0.362 0.331 11.42 0.379 0.346 9.75 0.324 0.295 7. Pork meat, fat 0.14 0.003 0.005 0.21 0.005 0.008 0.49 0.012 0.019 8. Pork meat, middle fat 0.93 0.020 0.026 1.07 0.023 0.030 1.83 0.039 0.051 F o r P e e r R e v i e w mgday -1 Total NaNO 3 intake 0.829 1.010 0.801 0.952 0.804 0.989	P99 0 0.020 0.464 0.429 0.195 0.911 0 0.057 0 0.160 0 2.236 0.105 0 0.015 0.592 0.229 0.305 1.359 0 0.031 0 0.107 0 0.127 2.575
			w w
			O n l O n l
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Food Additives and Contaminants

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Acknowledgements

Dietician Karin Hess Ygil, National Food Institute for incorporating data from sales statistics into the calculations.