Nitrite in feed: From Animal health to human health

Toxicology and Applied Pharmacology 270 (2013) 209–217

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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y t a a p

Contemporary Issues in Toxicology

Nitrite in feed: From Animal health to human health Andrew Cockburn a, Gianfranco Brambilla b, Maria-Luisa Fernández c, Davide Arcella d, Luisa R. Bordajandi e, Bruce Cottrill f, Carlos van Peteghem g, Jean-Lou Dorne e,⁎ a

Institute for Research on Environment and Sustainability, Devonshire Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NE17RU, UK Istituto Superiore di Sanità, Toxicological chemistry unit, Viale Regina Elena 299, 00161 Rome, Italy Departamento de Medio Ambiente, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Ministerio de Ciencia e Innovación, Carretera de la Coruña, 28040 Madrid, Spain d Unit on Data Collection and Exposure, European Food Safety Authority, Largo N. Palli 5/A43100 Parma, Italy e Unit on Contaminants in the Food chain, European Food Safety Authority, Largo N. Palli 5/A, 43100 Parma, Italy f Policy Delivery Group, Animal Health & Welfare, ADAS, Wolverhampton, UK g University of Gent, Harelbekestraat 72, 9000 Gent, Belgium b c

a r t i c l e

i n f o

Article history: Received 29 July 2010 Revised 29 October 2010 Accepted 15 November 2010 Available online 21 November 2010 Keywords: Risk assessment Nitrite Nitrate Feed Animal health Human health Toxicokinetics Toxicology Hazard identification Hazard characterisation Exposure assessment Risk characterisation

a b s t r a c t Nitrite is widely consumed from the diet by animals and humans. However the largest contribution to exposure results from the in vivo conversion of exogenously derived nitrate to nitrite. Because of its potential to cause to methaemoglobin (MetHb) formation at excessive levels of intake, nitrite is regulated in feed and water as an undesirable substance. Forages and contaminated water have been shown to contain high levels of nitrate and represent the largest contributor to nitrite exposure for food-producing animals. Interspecies differences in sensitivity to nitrite intoxication principally result from physiological and anatomical differences in nitrite handling. In the case of livestock both pigs and cattle are relatively susceptible. With pigs this is due to a combination of low levels of bacterial nitrite reductase and hence potential to reduce nitrite to ammonia as well as reduced capacity to detoxify MetHb back to haemoglobin (Hb) due to intrinsically low levels of MetHb reductase. In cattle the sensitivity is due to the potential for high dietary intake and high levels of rumen conversion of nitrate to nitrite, and an adaptable gut flora which at normal loadings shunts nitrite to ammonia for biosynthesis. However when this escape mechanism gets overloaded, nitrite builds up and can enter the blood stream resulting in methemoglobinemia. Looking at livestock case histories reported in the literature no-observed-effect levels of 3.3 mg/kg body weight (b.w.) per day for nitrite in pigs and cattle were estimated and related to the total daily nitrite intake that would result from complete feed at the EU maximum permissible level. This resulted in margins of safety of 9-fold and 5-fold for pigs and cattle, respectively. Recognising that the bulkiness of animal feed limits their consumption, these margins in conjunction with good agricultural practise were considered satisfactory for the protection of livestock health. A human health risk assessment was also carried out taking into account all direct and indirect sources of nitrite from the human diet, including carry-over of nitrite in animal-based products such as milk, eggs and meat products. Human exposure was then compared with the acceptable daily intake (ADI) for nitrite of 0-0.07 mg/kg b.w. per day. Overall, the low levels of nitrite in fresh animal products represented only 2.9% of the total daily dietary exposure and thus were not considered to raise concerns for human health. It is concluded that the potential health risk to animals from the consumption of feed or to man from eating fresh animal products containing nitrite, is very low. © 2010 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . Hazard identification and characterisation . . . . . Toxicokinetics of nitrite and nitrate . . . . . . Health effects in livestock species . . . . . . . Derivation of a health-based guidance value for

. . . . . . . . . . . . . . . . humans

⁎ Corresponding author. Fax: +39 0521 036 0472. E-mail address: [email protected] (J.-L. Dorne). 0041-008X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2010.11.008

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A. Cockburn et al. / Toxicology and Applied Pharmacology 270 (2013) 209–217

Occurrence of nitrite and nitrate in feed and water Exposure assessment in livestock . . . . . . . . Exposure assessment in humans . . . . . . . . . Risk characterisation for livestock and humans . . Livestock . . . . . . . . . . . . . . . . . . Humans . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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Introduction Nitrite, the anion of inorganic nitrite salts such as sodium nitrite, is formed naturally by the nitrogen cycle during the process of nitrogen fixation and is subsequently converted to nitrate, a major plant nutrient and constituent. Livestock feeding-stuffs contain both nitrite and nitrate and the latter is converted to nitrite and other metabolites (nitric oxide (NO) and N-nitroso compounds) either in the saliva of most monogastrics or in the fore-stomach/rumen of ruminants due to microbial activity. Adverse health effects in livestock and humans, resulting from acute and sub-acute exposure to excessive nitrite are typically due to the formation of MetHb in the blood. This can lead to cyanosis and at very high levels, death. The consequence of chronic exposure to nitrite is controversial with equivocal evidence of gastric carcinogenicity in female mice (Maekawa et al., 1987; NTP, 2001) but no clear evidence for direct carcinogenic potential in man. In order to protect animal and human health, the European Union, directive, 2002/32/EC on undesirable substances in animal feed, restricts the maximum content of nitrite in complete feedingstuff (with a moisture content of 12%) for livestock excluding birds and aquarium fish to 15 mg/kg, and the maximum content of fish meal to 60 mg/kg. The current review highlights, the recent risk assessment performed by the scientific panel on contaminants in the food chain (CONTAM Panel) of the European Food Safety Authority (EFSA) regarding the impact of nitrite in animal feed for livestock health and human health following the consumption of animal products such as milk, meat and eggs, (EFSA, 2009). Particular focus is given to toxicokinetic and toxicological aspects in food producing animals, laboratory animals and humans to help explain the potential health impacts of dietary nitrite. Finally, a risk characterisation comparing exposure scenarios in animals and humans and safe levels of exposure concludes the review. Hazard identification and characterisation Toxicokinetics of nitrite and nitrate The toxicokinetics of nitrate have been reviewed elsewhere in detail for non-ruminant and ruminant livestock species, laboratory and companion animals and humans (EFSA, 2008, 2009). Interspecies differences in toxicokinetics for nitrite and nitrate provide a valuable physiological basis to identify potentially susceptible species and populations to the toxicity of both anions. Such interspecies differences are illustrated in Fig. 1 for pigs, cows and humans. The oral rate of absorption of nitrite and nitrate is low in nonruminants (pigs) and ruminants since minor amounts (10–20%) pass from the stomach and the rumen respectively to the blood stream as nitrite (EFSA, 2009). In contrast, oral absorption of both nitrite and nitrate is high in rodents and humans (90–95%) (Kortboyer et al., 1997). After absorption, nitrite is rapidly distributed in the plasma and binds to erythrocytes. Interspecies differences in volume of distribution of nitrite have been documented with 1624, 278 and 192 ml/kg body weight (b.w.) in the dog, sheep and pony, respectively, after intravenous administration of 20 mg/kg sodium nitrite b.w. (Schneider and Yeary, 1975). In contrast to nitrite, interspecies differences in the

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. 213 0 . 214 0 . 214 0 . 215 0 . 215 0 . 215 0 . 215 0 . 216 0 . 216 0

volume of of distribution for nitrate are low ranging between 210 and 330 ml/kg b.w. in humans, ponies, sheep and goats (Schneider and Yeary, 1975; Schultz et al., 1985; Lewicki et al., 1998; EFSA, 2008). After binding to the erythrocyte s membrane, nitrite is reduced to NO (by xanthine oxidase and NO synthase) which has a wide range of physiological functions in health and disease as a second messenger (Lundberg et al., 2008; Webb et al., 2008). In monogastrics (humans, dogs and mini-pigs), 5–7% of the absorbed nitrate is concentrated from the plasma to the saliva through an entero-salivary recirculation pathway and reduced to nitrite by the nitrite reductase from commensal bacteria present on the back tongue. Approximately 20% of the salivary nitrite is then swallowed into the stomach where it is reduced to NO, oxidised to nitrate in the plasma and re-circulated through the entero-salivary circulation (EFSA, 2008). In contrast, under the acidic conditions of most monogatrics stomachs (pH b 3.5) nitrate is metabolised to nitrous acid which in turn spontaneously decomposes to nitrogen oxides including NO (Wright and Davison, 1964; Mirvish, 1975). Endogenous production of NO occurs in the urea cycle using L-arginine as a substrate and NO synthase. However, it has been estimated that exogenous intake of nitrite/nitrate leads to NO levels, in the upper intestine, up to 10,000 times higher than that from endogenous production (McKnight et al., 1997). Conditions of excessive plasma nitrate result in reduction to nitrite; nitrite reacts with Hb to produce MetHb which can be reduced back to Hb via MetHb reductase (Fig. 1). Physiological levels of MetHb in the human blood range between 1 and 3%, and reduced oxygen transport has been noted clinically when MetHb concentrations are above 10% (Walker, 1990; FAO/WHO, 2003a,b). Cyanosis and hypoxia occurs above 20%, and levels above 50% can be life threatening (Mensinga et al., 2003). Infants under 3 months of age are more susceptible to MetHb due a 40– 50% lower MetHb reductase levels compared with adults. Moreover, in foetuses and neonates, Hb has a higher affinity for oxygen and hence forms MetHb more readily than adults (WHO, 1997). Finally, because of a relatively high gastric pH, infants have an increased likelihood of intestinal infections where pathogenic bacteria can rapidly reduce nitrate to nitrite (Savino et al., 2006). Interspecies variability in MetHb reductase activity has also been estimated, as a percentage of human activity and partially accounts for differences in sensitivity to MetHb between species. Pigs have been shown to lower MetHb reductase (27%) compared with horses (63%), cattle, cats and goats (90%), dogs (114%), sheep (150%) and rabbit (452%). Such low MetHb reductase in pigs together with relatively low nitrite reductase levels in the saliva provide a metabolic rationale for their physiological sensitivity to nitrite toxicity. Intra-species differences in MetHb reductase activity have also been shown to be associated with congenital defects as well as age-related differences in reductase expression between neonates and older animals (Harvey, 2006). In contrast to pigs, the rumen and enlarged caecum of cows (in addition to their relatively high pH (N5)) are especially well suited for nitrate reductase activity. This results in only10–20% of nitrite absorption into the blood stream as the bulk is metabolised by rapidly adaptable gut flora to ammonia for onward synthesis into amino acids and protein or eliminated with other gases during eructation (Lewis,

A. Cockburn et al. / Toxicology and Applied Pharmacology 270 (2013) 209–217

Pigs

Humans

NO 3 /NO2

Low absorption stomach

-

Mouth: Low bacterial nitrite reductase

Mouth: Bacterial nitrite reductase

High absorption stomach Entero-salivary circulation

Salivary uptake

Plasma

NO 3

-

NO 3 /NO 2

Entero-salivary circulation

Urinary excretion

211

Salivary uptake

Salivary NO2-

Urinary excretion

Plasma NO 3-

Salivary NO2-

Acidic stomach

Acidic stomach

NO Low MetHb reductase

Hb

NO MetHb reductase (Low in neonates)

Plasma

MetHb

Hb

-

MetHb

Plasma NO3

Plasma

Plasma NO2Urinary excretion

Toxicity

Plasma

-

Plasma NO3

NO 2-

Toxicity

Urinary excretion

Cows -/

-

NO3 NO2

Low absorption Forestomach NO2

Rumen Bacterial reductase

MetHb reductase

Hb

-

PlasmaNO3

MetHb

NH3

Plasma NO 2-

Rumen

Toxicity

Amino acid and protein synthesis

Legend: NO3-/ NO 2-: oral nitrate/nitrite exposure through feed (pigs and ruminants) and food (humans); Plasma NO3-: plasma nitrate, Plasma NO 2-: plasma nitrite; salivary NO 2-: salivary nitrite; NO: nitric oxide; Hb: hemoglobin; MetHb: methemoglobin; MetHb reductase: methemoglobin reductase. Bold characters and Dashed lines indicate susceptibility factors to toxicity in pigs (reduced bacterial reductase in the mouth and low MetHb reductase activity), humans (low MetHb reductase in neonates) and cattle (overwhelming of the rumen adapation leading to methemoglobin formation) Fig. 1. Interspecies differences in the metabolism of nitrite and nitrate.

1951; Wang et al., 1961; Winter, 1962; Wright and Davison, 1964; Sen et al., 1969; Mirvish et al., 1975) (Lewicki et al., 1998; Witter et al., 1979a,b; Hartman, 1982). This rumen conversion is an adaptive process depending on the nitrite/nitrate content in the rumen which normally has the capability of responding to widely varying loadings. However, excessive intakes of nitrite/nitrate in cows can result from the large daily intake of water (growing cattle can consume 30/60 L/ day) and feed. Drinking water can potentially contain both high nitrate levels and significant coliform contamination (N10 CFU/100 mL) able to reduce nitrate to nitrite. Feed sources can include silages and hay (N200 mg/kg nitrite) which may represent 50% or more of the dry feed intake and milk replacers fed to calves. Such intakes can overwhelm the rumen pathway resulting in nitrite accumulation faster than its

conversion to ammonia and the potential for MetHb formation. As described for pigs and humans, the excess nitrite then reacts with Hb to potentially form MetHb (Bruning-Fann and Kaneene, 1993; Baranova et al., 2000). In contrast with adult cows, calves do not possess a rumen (monogastrics) and their susceptibility to nitrite is due to low MetHb reducaste activity as observed in pigs (EFSA, 2009). Excretion of nitrite is rapid and extensive in the urine with no accumulation in tissues with short elimination half-lives below an hour (30 min in the dog, sheep and pony and around 40 min in humans) (Schneider and Yeary, 1975; Dejam et al., 2007). Nitrate excretion is slower with over 80% of urinary nitrate pumped back into the blood stream by active transport but it is maximal after 5 hours and complete within 18 hours (Walker, 1996).

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A. Cockburn et al. / Toxicology and Applied Pharmacology 270 (2013) 209–217

Health effects in livestock species MetHb intoxication can result from a variety of interspecies differences vide supra and exposure scenarios (as described above), together with other factors such as inter-current treatment with antibiotics which can impact both on the salivary and gastrointestinal flora. Despite the potential risk of nitrite poisoning, adverse effects in livestock are relatively uncommon due to farmer awareness of the predisposing factors leading to resultant precautionary husbandry practices. Clinical signs of acute nitrite toxicity in a range of livestock associated with MetHb are generally dose-dependent due to oxygen starvation and may include accelerated pulse, dyspnoea, muscle tremors, weakness, vomiting, unstable gait, and cyanosis leading to death. Symptoms of sub-chronic and chronic toxicity include reduction in feed intake, milk production in dairy animals, rough hair and reduced weight gain or actual loss. In pregnant animals, low fertility and abortion, the latter correlated with foetal hypoxia due to MetHb, can also occur. Nitrite has also been reported to cause goitrogenicity in poultry whereas nitrate has been shown to have this effect in swine, sheep and cattle resulting from inhibition of iodine uptake from the thyroid gland by NO (Bazzara et al., 2007). The literature and the corresponding database regarding the toxicity of nitrite and nitrate present in feed is limited for livestock species. For the purpose of risk assessment, NOAELs and LOAELs for a number of species have been derived and are illustrated in Table 1; pending a number of limitations and uncertainties which relate to data quality and availability: Unlike well-controlled laboratory animal studies, the data reported from livestock are usually derived from a wide variety of husbandry practices, dietary and dosage regimes (eg complete feeding-stuffs, direct dosing with sodium nitrite or nitrate, co-exposure to nitrate and nitrite, aggregate exposures from soil, water, feed and forages). -In the absence of toxicity data for nitrite in livestock, NOAEL estimates were either derived using published data on nitrate, converted to nitrite, based on evidence for a 10-fold ratio in acute toxicity, or derived from LOAELs from exposures causing minimal adverse effects using an uncertainty factor of 3. For cases where LD50 data were the only figures

available, LOAELs for nitrite or nitrate toxicity were estimated to be 10% of the LD50 value (EFSA, 2009). Generally speaking the major daily feed rations of most monogastric species are grain and cereals, which naturally have very low nitrite levels and thus contaminated water can prove to be a greater problem than feed. In pigs, lethality has been reported after a single oral dose of sodium nitrite above 20 mg/kg b.w. per day (Muirhead and Alexander, 1997). No toxicity data relating to nitrite exposure were available for horses (caecal-colonics), however MetHb formation and death associated with MetHb levels of 70% were observed after single doses of 100 and 200 mg/kg nitrate b.w. per day respectively (Bradley et al., 1940). In contrast, MetHb formation in New Zealand White Rabbits was observed following acute oral dosage of 88 mg/kg b.w. per day nitrite confirming their lower sensitivity due to their relatively high levels of MetHb reductase. Poultry are mostly fed cereal/grain based feeds and thus nitrite poisoning is rare. For ruminants, while no specific reports of nitrite exposure and toxicity are available for adult cattle, intoxications and MetHb formation have been reported and result from nitrate exposure with LD50 estimates of 330 and 990 mg/kg b.w administered via drenching; or feed respectively (Bradley et al., 1940). Toxicity data in dairy cows refer to nitrate or combined exposure from nitrite/nitrate with subacute abortion observed at nitrite levels of 3000 mg/kg between 2– 21 days following exposure to DM in feed corresponding to approximately 115 mg/kg b.w. per day (calculated using a body weight of 625 kg and a feed intake of 25 kg DM). Exposure of calves to aqueous solutions of nitrite and nitrate corresponding to 34 mg/kg b.w. and 244 mg/kg b.w. per day respectively led to MetHb and cyanosis (Baranova et al., 2000). In Sheep, oral lethal doses for sodium nitrite were in the range of 67–110 and 83 mg/kg b.w. day (Bartik and Piskac, 1981; Trif et al.;, 1993). Mild MetHb (10%) was observed at 50 mg/kg b.w. 4 hours after administration whereas the same dose administered over 7 consecutive days did not induce clinical signs supporting adaptation of the rumen flora to high nitrite/nitrate exposure from a sub-acute chronic perspective (Trif et al., 1993).

Table 1 LOAELs and estimated NOAELs derived from the lowest exposure to nitrite (or nitrate) reported to induce toxicity in livestock and companion species. Species

Cattlea

Substrate

Calves

Nitrate in feed (estimated to nitrite) Nitrite per se

Sheep

Nitrite per se

Horsesa

Nitrate in feed (estimated to nitrite)

Growing Pigs

Nitrite in feed

Sows

Nitrite per se

Rabbits

Nitrite per se

Poultry

Nitrite per se

Cats

b

Nitrite in food

Dogs

Nitrite per se

Fish (trout)c

Nitrite per se

Toxicity endpoint

LOAEL

NOAEL

(references)

mg/kg b.w. per day

mg/kg b.w. per day

LD50 MetHb Bradley et al. (1940) LOAEL Baranova et al. (2000) NOAEL Trif et al. (1993) LD50 MetHb Bradley et al. (1940). LOAEL Koch et al. (1963) Seerley et al. (1965) Lack of developmental defects NOEL Sleight et al. (1972) Urinary hormone excretion changes Violante et al. (1973) Liver and kidney function Sell and Roberts (1963) MetHb FAO/WHO, 1974 MetHb Michalski (1963) Met Hb

Methaemoglobinemia.1 a Estimated from feed exposure to nitrite using a 10:1 ratio for nitrate:nitrite ratio. b Data referred to one animal. c Data reported on water in mg/L, MetHb.

9.9 34

3.3 11 10

10

3.3

10

3.3

–

17.2

13.4

4.5

75

25

69

23

7.9 –

2.6 0.1

A. Cockburn et al. / Toxicology and Applied Pharmacology 270 (2013) 209–217

Based on the limited data presented in Table 1, the most sensitive species were identified as the pig, the cow and the calf with NOAEL values of 3.3, 3.3 and 11 mg/kg b.w per day. Toxicity in laboratory animals and relevance to human health. Nitrite has been studied extensively over the last fifty years. The principal adverse effect consistently observed in livestock, rodents and man is methemoglobinemia. A full range of toxicity studies can be found in the literature spanning acute, sub-acute and chronic as well as genotoxicity and somewhat limited reproductive toxicology. Not all of studies were conducted according to modern day standards but the results are sufficiently consistent to give confidence for the establishment of the toxicological profile. The acute toxicity of nitrite is approximately 10-fold higher than that of nitrate. LD50 values (mg/kg b.w) for sodium nitrite and sodium nitrate were available from the literature for mice (214 vs 2500–6500), rats (180 vs 3300–9000) and rabbits (186 vs 1900–2680) (corresponding for the anion nitrite vs the anion nitrate to 143 vs 1525–3810 in mice, 121 vs 2440–6660 in rats and 126 vs 1410–2000 in rabbits) (NIOSH, 1987; Speijers et al., 1987; Walker, 1990). Sub-acute exposure of rats to sodium nitrite resulted in hypertrophy of the adrenal zona glomerulosa with a NOAEL of 5.4 mg/kg b.w. per day (Til et al., 1997; Boink et al., 1998; Mensinga et al., 2003). This finding was considered unlikely to be of relevance to livestock or human health because of the relatively high dose levels involved and the fact that the NOAEL was significantly higher than the levels of nitrite to which livestock or man are typically exposed. Studies on chronic toxicity of sodium nitrite from drinking water intake in rats and mice were performed under the National Toxicology Programme (NTP, 2001). Daily doses of sodium nitrite to mice were equivalent to 0, 60, 129 or 220 mg/kg b.w. per day for males (equivalent to 40, 80 and 147 mg/kg nitrite ion b.w. per day), and 0, 45, 90 or 165 mg/kg b.w. per day for females (equivalent to 0, 30, 60, 111 mg/kg nitrite ion b.w per day). The NTP concluded that these studies provide equivocal evidence of the carcinogenic activity of nitrite (“studies that are interpreted as showing a marginal increase of neoplasms that may be chemical related”). In males, the incidence of hyperplasia of the glandular stomach epithelium was significantly greater at the highest dose and the authors concluded that there was equivocal evidence for carcinogenic activity in females based on the trend in the combined incidence of squamous cell papilloma and carcinoma of the fore stomach (NTP, 2001). Derivation of a health-based guidance value for humans Nitrite and nitrate were first evaluated in 1961 for risks associated with ingestion at the second meeting of the Joint Food and Agriculture Organisation/World Health Organisation (FAO/WHO) Expert Committee on Food Additives (JECFA) (FAO/WHO, 1962). A number of reviews were subsequently undertaken by the former Scientific Committee on Food of the European Commission (SCF) in 1990, and 1995, the JECFA in 1995 and 2002, and EFSA in 2008 to establish health-based guidance values as Acceptable Daily Intakes (ADI) for both ions (EC, 1992, 1997; FAO/WHO, 1995, 2003a,b; EFSA, 2008). In 1995, the SCF derived an ADI of 0–0.06 mg/kg for nitrite (SCF, 1997). In 2002, the JECFA derived an ADI for nitrite of 0–0.1 mg/kg b.w. per day for the sodium salt and 0–0.07 mg/kg b.w. per day for the anion (FAO/WHO, 2003a,b). In 2008, this value also was endorsed by the CONTAM Panel of EFSA because of no significant new toxicological and toxicokinetic data (EFSA, 2008). The ADI for nitrite was derived using NOAELs for sodium nitrite and the nitrite ion of 10 and 6.7 mg/kg b.w. per day, respectively, and applying an uncertainty factor of 100. The identification of the NOAEL of 10 mg/kg b.w per day was based on a 2year oral study in rats using pulmonary toxicity (dilatation of the bronchi) and cardiac toxicity (focal degeneration and fibrosis of the heart muscle, dilatation of coronary arteries with infiltration of lymphocytes and emphysema at the highest dose) (Maekawa et al., 1982).

213

The ADI for sodium nitrate (5 mg/kg b.w. per day) or the anion (3.7 mg/kg b.w. per day) have been derived by the JECFA, the SCF and EFSA. NOEL values of 500 mg/kg b.w. per day for sodium nitrate and and 370 mg/kg b.w. per day nitrate ion respectively from a subchronic study in dogs (125 days) and a chronic study in rats using growth retardation as the toxicological endpoint and by applying an uncertainty factor of 100 (FAO/WHO, 1962; SCF, 1992, 1997; Lehman, 1958 cited in FAO/WHO, 1962; Lijinsky et al., 1973; EFSA, 2008). It has been argued that the rat may not be a good model for humans because of its low conversion of nitrate into nitrite in the saliva. However, because of the importance of the chronic toxicology, the rodent toxicokinetics and similar NOELs found in the dog (a relevant model for humans) these studies continue to be considered to be relevant for risk assessment (EFSA, 2008).

Occurrence of nitrite and nitrate in feed and water The main source of dietary nitrite results from that naturally present as nitrite/nitrate in feeding-stuffs or less commonly that which has been added as a preservative, for example sodium nitrite in the production of silage. Contaminated drinking water can also be an important source. The natural levels of nitrite in fresh plant material is with certain exceptions, generally very low (Trif et al., 1986; EFSA, 2008) and as noted by the Scientific Committee on Animal Nutrition (SCAN) of the European Union (EC, 2003) typical levels in feeding-stuffs have not been reported to cause toxicity in farm animals. Nitrite is not normally present in soils to any significant extent, and as a result it is not normally available for uptake by plants. As in the case of vegetables grown for human consumption there are exceptions regarding low levels of nitrate in plant material and nitrate poisoning has been associated with more than 80 forage species (Clarke and Clarke, 1975). Practically, the majority of these species do not represent major feeds for livestock. Nitrate concentration for common feeds that are considered to be safe range from 4 to 1760 mg/kg in soybean meal and fresh alfalfa or alfalfa hay with intermediate values of 22, 44 and 880 mg/kg in maize grain, oat grain and alfalfa silage, respectively (Crowley, 1985). A range of factors in addition to the plant species can affect the nitrate contents of common feeds and these include the impact of fertilizer application rates, growing conditions (higher nitrate levels are associated with poor growth) and the stage of maturity (young plants contain more nitrate) (Cooper and Johnson, 1984; Osweiler et al., 1985; Gupta, 2007). When taking into account different fertilizer or harvesting regimens, nitrate concentrations in forages (in mg/kg DM) would raise from 1800 to 3200 mg/kg from the first to the second vegetative alfalfa crop, 4400 mg/kg in fresh chopped maize, 868 to 2627 mg/kg for barley at soft dough (67 and 134 kg/Ha) and 2149 to 5613 mg/kg for oat hay soft dough (67 and 134 kg/Ha) (Cash et al., 2007). Fresh forage crops such as maize, grasses, legumes, wheat and lucerne can be preserved by ensiling and are highly valued as animal feed. In order to preserve the crops successfully it is important to achieve good microbial fermentation. Frequently a chemical or microbial additive is applied to the crop at harvesting for this purpose. Sodium nitrite is used to restrict the activity of undesirable bacteria which compete with beneficial bacteria for nutrients while at the same time reducing the quality of the protein in the forage (Woolford, 1978). Sodium nitrite was used as a preservative in European Fisheries in the l940's because it was more effective than salt. In the l960's serious health effects began to be recognised in livestock fed fish meal (Sakshaug et al., 1965; Koppang, 1974). The toxicant was identified as N-nitrosodimethylamine (NDMA) which has been shown to be carcinogenic and which was associated with an interaction between the nitrite and amino acids. In consequence an alternative method of preserving fish was developed and since the 1990s sodium nitrite as fish meal is prohibited.

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Table 2 Total levels of nitrite in mg/kg reported by country for main feed commodities in comparison with maximum limits. Feed commodity group

Country

ML Nitrite (mg/kg)

a

Complementary feed Complete feed Fish — complete feed Fishmeal

Forage Other feed Total

Slovenia Slovenia Cyprus Cyprus France Slovenia Slovenia Slovenia

– 10 – 40 40 40 – –

Mean (mg/kg)

Median (mg/kg)

Maximum (mg/kg)

0.7 2.5 0.3 1.6 1.4 2.5 6.7 2.6

0.7 1.6 0.3 0.3 1.0 2.5 3.5 1.7

0.7 7.9 0.3 11.3 11.2 2.5 26.2 6.5

Samples No

N LODs or LOQs

NMLs

3 15 13 17 27 3 7 9 94

0 4 0 4 8 0 4 3 23

– 0 – 0 0 0 – – 0

MLs: maximum limit for nitrite in feed (mg/kg); mean; mean value for nitrite in feed (mg/kg); median: median value for nitrite in feed (mg/kg); maximum: maximum value for nitrite in feed (mg/kg); no: number of samples analysed for nitrite in feed (moisture content of 12%) in the period 2002–2008. NLOD or LOQs: number of samples that exceeded the limit of detection (LOD) or limit of quantification (LOQ); N MLs: number of samples that exceeded maximum limits (MLs). a Complementary feeding stuff: a mixture with a high content of certain substances, which, because of their composition, is only sufficient for a daily ration if used in combination with other feeding stuffs.

On farms, surface and well waters can be consumed by livestock in addition to piped drinking water from a mains supply. This can represent in a significant source of nitrate exposure as it is extremely soluble and can easily pass through soil from manures, fertilizers and other wastes such as excreta. According to an European Commission Report (COM 120, 2007) on the implementation of Council Directive 91/676/EEC regarding the protection of waters from nitrate pollution from agricultural sources for the period 2002–2003, it is not unusual for nitrate levels to exceed 100 mg/L which is 200 times the upper EU limit set by the Drinking Water Directive of 0.5 mg/L. In order to relate published data with typical nitrite/nitrate levels in animal feed to the practical situation in Europe, three member States (Cyprus, France and Slovenia) submitted nitrite concentrations in feedingstuffs to EFSA for the period 2002–2008. These values were acquired during their routine surveillance programmes measured using either the colourimetric method or spectrophotometry. Levels of nitrite were available for 94 samples of feed were received from 3 European countries. Slovenia was the only country to additionally provide data on levels of sodium nitrate in 22 samples of feed for the period 2003–2007. Mean, median and maximum values for the different types of feed were 31.3, 31.3 and 47.3 mg/kg for complementary feed. 8.6, 7.9 and 19.6 mg/kg for complete feed, 58.4, 1.8 and 394.1 mg/kg forgae and 13.0, 1.8 and 40.6 mg/kg other feeds (EFSA, 2009). Table 2 provides summary statistics (mean, median and maximum values), for 3 EU countries for each of the main feed commodity groups for nitrite. In the same tables, information on the number of samples that exceeded the limit of detection (LOD) or of quantification (LOQ) and, in the case of nitrite, above the permitted Maximum Limits (MLs), was also reported. Only 23 samples out of 94 exceeded the LODs or LOQs, no samples above the MLs for nitrite were detected. The highest levels of nitrite and nitrate were detected in fodder in Slovenia, 6.7 and 58.4 mg/kg on average, respectively.

Exposure assessment in livestock Using the EU maximum permitted levels of exposure to nitrite in complete feed (mg/kg) together with the maximum limit of 0.5 mg/L for water, and allowing for estimates of feed and water consumption by livestock typical within Europe, levels of nitrite exposure have been estimated for monogastric livestock and are shown in Table 3a. A similar exercise was conducted for ruminant exposure to nitrite in compound feed containing the maximum permitted nitrite concentration (10 mg/kg), forage at the maximum value reported by Slovenia (26.2 mg/kg) (Table 2), and water at the EU maximum limit value for nitrite (0.5 mg/L) as shown in Table 3b. Overall, the results show that at maximum permitted levels of nitrite in feeding-stuffs exposure could be 0.37 mg/kg nitrite b.w. per day in pigs (Table 3a) and 0.65 mg/kg nitrite b.w. per day in cattle (Table 3b). If the contribution from drinking water is taken into account, the exposure would rise to 0.42 and 0.70 mg/kg nitrite b.w. per day in pigs and cattle, respectively. A small but significant exposure to nitrite can also occur in grazing animals due to soil ingestion and such exposure has been estimated to vary between 1 and 18% of the feed DM intake (Thornton and Abrahams, 1983). Exposure assessment in humans Potential exposure of consumers to nitrite contained in fresh animal products could theoretically occur if significant carry-over into the human diet were to take place via the consumption of fresh milk, meat and eggs. However, nitrite levels have not been reported in milk or at detectable levels in egg samples (Ologhobo et al., 1996). Moreover, only trace levels were found in the meat from slaughtered pigs (Eleftheriadou et al., 2002). In the UK, nitrite dietary exposure estimated by means of a total diet study, represents on average

Table 3a Estimated exposure of monogastric livestock to nitrite from feed and water given a diet containing the maximum permitted sodium nitrite concentration (15 mg/kg) expressed as nitrite ion (10 mg/kg) and water at the EU maximum limit value (EFSA, 2009). Species

Pigs Sows Poultry (broilers) Poultry (laying hens) Fish 1

Live weight

Consumption Total complete feed

Water

Nitrite intake from Total complete feed1

kg

kg/day

l/day

mg/kg b.w. per day

100 250 2.1 1.9 4.5

3.7 6.5 0.15 0.115 0.09

10 25 0.02 0.02 30

0.37 0.26 0.71 0.61 0.20

Nitrite in total complete feed assumed equal to 10 mg/kg;2 Water contribution using the EU maximum limit value of 0.5 mg/L is given for comparative purposes.

Water2

0.05 0.05 0.00 0.001 3.33

A. Cockburn et al. / Toxicology and Applied Pharmacology 270 (2013) 209–217

215

Table 3b Estimated exposure of ruminants to nitrite from feed and water where the diet contains the maximum permitted sodium nitrite concentration (15 mg/kg) in compound feed and the maximum reported concentration in forage, expressed as nitrite. These calculations assume typical livestock feed and represent a worst-case scenario (EFSA, 2009). Species

Dairy cow Suckler cow Cattle Lactating ewe Growing lamb Dairy goats

Live weight

Consumption

Nitrite intake from

Forages1

Compound feed2

Water

Forages3

Kg

kg/day

kg/day

l/day

mg/kg b.w. per day

625 550 300 70 20 65

14.0 11.0 7.0 1.5 0.45 1.50

10.0. 5.0. 1.0 0.3 0.15 0.7

120 60 30 15 5 15

0.59 0.52 0.61 0.56 0.59 0.60

Compound feed4

Water5

0.18 0.10 0.04 0.05 0.09 0.12

0.10 0.05 0.05 0.11 0.13 0.12

ML = maximum limit; DM = dry matter; 158% of the diet DM; 242% of the diet DM; 3Nitrite intake from forages using the maximum value of 26.2 mg/kg DM reported by Slovenia; 4 Nitrite intake from compound feed, using 11.36 mg nitrite/kg DM, (10.0 mg/kg for a feed with a moisture content of 12%); 5Nitrite in water assumed equal to 0.5 mg/l.

1.5 mg/person per day (EFSA, 2008). Fresh meat represents only 4% of the total intake which along with other fresh animal products, milk and dairy products (6%), eggs (3%) and fish (1%) combines to a total of 14% of the total daily nitrite intake. Other contributors include preserved meat products (19%), vegetables and fruit (15%) and water (7%). The remaining 47% comes from other foods such as bread, cereals, oil and fats, sugar preserves, beverages and nuts (MAFF, 1998). Nitrate exposure is much larger (91 mg/person per day) and is mostly through the consumption of vegetables, water, beer and other foods. Fresh animal products only represent a minor fraction of the total exposure, around 7%. In comparison to nitrite intake, bioconversion of dietary nitrate to nitrite represents some 82% of the total daily nitrite intake. In contrast, direct exogenous nitrite intake from the diet constitutes less than 20% of the overall combined total daily nitrite exposure, which is some 7.3 mg nitrite/person per day. Taking into account the contribution of nitrite from fresh animal based products alone, this represents as little as 2.9% of the total combined daily nitrite exposure. Risk characterisation for livestock and humans The main potential hazard for livestock and man from acute nitrite ingestion (or nitrite resulting from inter conversion from nitrate is methaemaglobinaemia and its associated adverse effects. This potential is only realised at relatively high levels of exposure and the following section sets out to examine the likelihood for methaemaglobinaemia to occur in livestock husbanded under good agricultural practise or in humans receiving fresh animal products (milk, meat and eggs) from such animals. Livestock Based on the scientific literature, the derived NOAELs for nitrite exposure for a range of vertebrate species, and with the notable exception of fish, were found to be relatively comparable and to fall within an order of magnitude. For risk characterisation, pigs and adult cattle represented the more sensitive livestock species with an estimated NOAEL for both species of 3.3 mg/kg b.w. per day. The potential “worst-case” nitrite intake for both species was also estimated using the maximum permitted level in complete feed under the current legislation (10 mg/kg), in water (0.5 mg/L) and also the maximum level found in forages from a Member State (Slovenia, 26.2 mg/kg). In this scenario which used “worst-case” considerations for nitrite exposure, overall nitrite intake from feed was 0.37 and 0.65 mg/kg b.w. per day in pigs and cattle which when compared with the estimated NOAEL of 3.3 mg/kg b.w per day for both species resulted in margins of safety (MOS) of 9 and 5, respectively. Good farming practise combined with farmers awareness of the risk of nitrite intoxication in livestock from feeding-stuffs such as forages, silages, hay and contaminated drinking water, means that these margins of safety are considered adequate to protect animal health.

Humans Overall data on the carry-over and residues of nitrite in animal tissues and animal products are very scarce. However, because of the rapid excretion of nitrite, the likelihood of accumulation in animal tissues and products such as milk and eggs is low. The impact of nitrate loading on milk quality in cows 2 hours before the evening milking of dairy cows has been investigated experimentally. 9.5, 18.75, 37.5, 75 and 150 g of potassium nitrate were administered to cows before milking and nitrate residues were quantified in individual milk samples 2, 14, 26, 38 and 50 h after nitrate loading with average concentrations collected after 2 h time point of 3.4, 4.5, 9.8, 15.6 and 34.6 mg nitrate/L (Baranova et al., 1993). Thus the carry-over was not found to be strictly dose related. Eleftheriadou et al. (2002) analysed nitrite and nitrate levels from 120 muscle samples from 5 pig breeds. In addition 20 feed and 20 drinking water samples were analysed. Only trace amounts of nitrite and low concentrations of nitrate were found in the samples (7.5–15.7 mg nitrate/kg in meat, 9.3–13.4 mg nitrate/kg in animal feed and from 28.0 and 65.2 mg nitrate/L in the water). There was no correlation between the nitrate in feed and water and its concentration in the muscle samples collected. From this, together with the toxicokinetic profile for nitrite, it can be concluded that human exposure from carry-over of residues in livestock products (fresh milk, meat and eggs) is likely to be low and that nitrite does not readily accumulate in animals. Such a small proportion of the total daily intake of nitrite coming from fresh animal products does not raise any concern for human health. Conclusions This review focused on a recent risk assessment investigating the health impact of the presence of nitrite in animal feed for livestock species and humans consuming animal products (EFSA, 2009). Plantbased animal feed naturally contains low levels of nitrite based on its vegetable content together with contributions from hay and silage and also from sodium nitrite, sometimes added directly as a preservative. Under normal manufacturing/husbandry practise the majority of the feed produced in Europe contains nitrite well below the EU maximum limits based on limited but recent evidence from three EU Member States. Notwithstanding, the majority of nitrite exposure in livestock results from the endogenous inter-conversion of nitrate to nitrite in feed and water. The main adverse effect in humans and livestock related to nitrite exposure is the formation of MetHb, which can be reversed at low levels, by an age and species dependent reductase. Amongst food producing livestock, pigs, cows and calves were identified as the more sensitive species from a limited toxicological database. Pigs are sensitive to nitrite due to their relatively low nitrite and MetHb reductase activity. Calves, which are monogastric as juveniles, also have a low MetHb reductase activity. Cows have a large daily food and water intake and potentially highly adaptable rumen flora. However, excessive nitrite/nitrate intake

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can overwhelm the normal the process of anabolism to ammonia, resulting in the potential for significant systemic exposure to nitrite and MetHb formation. NOAELs for nitrite intake for pigs and cows have been estimated from the literature at approximately 3.3 mg/kg b.w. per day for both species. The total nitrite intake for pigs and cattle consuming complete feed containing nitrite at the maximum limit under EU legislation was found to be 9-fold and 5-fold lower than the NOAEL. Based on the bulkiness of animal feed this represents a satisfactory margin of safety to protect animal health and welfare. Despite the very limited quantitative information on nitrite residues in fresh animal products the evidence indicates that the potential for accumulation of nitrite in animal tissues is very low. This is due to its rapid metabolic turnover and excretion. Separate calculations, based on dietary surveys conducted in EU Member States, showed that compared with the total daily exposure to nitrite, most of nitrite exposure derives from interconversion from nitrate contained in fruit and vegetables, fresh animal products contribute a maximum of 2.9% of the total daily exposure to nitrite. It is concluded that such a contribution of nitrite from animal products does not raise any concern for human health.

Conflict of interest No competing interests between the authors were identified.

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