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 Toxicology and Applied Pharmacology 270 (2013) 209–217

Contents lists available at ScienceDirect

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
b
Istituto Superiore di Sanità, Toxicological chemistry unit, Viale Regina Elena 299, 00161 Rome, Italy
c
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

a r t i c l e i n f o a b s t r a c t

Article history: Nitrite is widely consumed from the diet by animals and humans. However the largest contribution to
Received 29 July 2010 exposure results from the in vivo conversion of exogenously derived nitrate to nitrite. Because of its potential
Revised 29 October 2010 to cause to methaemoglobin (MetHb) formation at excessive levels of intake, nitrite is regulated in feed and
Accepted 15 November 2010
water as an undesirable substance. Forages and contaminated water have been shown to contain high levels
Available online 21 November 2010
of nitrate and represent the largest contributor to nitrite exposure for food-producing animals. Interspecies
Keywords:
differences in sensitivity to nitrite intoxication principally result from physiological and anatomical
Risk assessment differences in nitrite handling. In the case of livestock both pigs and cattle are relatively susceptible. With
Nitrite pigs this is due to a combination of low levels of bacterial nitrite reductase and hence potential to reduce
Nitrate nitrite to ammonia as well as reduced capacity to detoxify MetHb back to haemoglobin (Hb) due to
Feed intrinsically low levels of MetHb reductase. In cattle the sensitivity is due to the potential for high dietary
Animal health intake and high levels of rumen conversion of nitrate to nitrite, and an adaptable gut flora which at normal
Human health loadings shunts nitrite to ammonia for biosynthesis. However when this escape mechanism gets overloaded,
Toxicokinetics
nitrite builds up and can enter the blood stream resulting in methemoglobinemia. Looking at livestock case
Toxicology
histories reported in the literature no-observed-effect levels of 3.3 mg/kg body weight (b.w.) per day for
Hazard identification
Hazard characterisation nitrite in pigs and cattle were estimated and related to the total daily nitrite intake that would result from
Exposure assessment complete feed at the EU maximum permissible level. This resulted in margins of safety of 9-fold and 5-fold for
Risk characterisation 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
0
Hazard identification and characterisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
0
Toxicokinetics of nitrite and nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
0
Health effects in livestock species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
0
Derivation of a health-based guidance value for humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
0

⁎ Corresponding author. Fax: +39 0521 036 0472.

E-mail address: jean-lou.dorne@efsa.europa.eu (J.-L. Dorne).

0041-008X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2010.11.008
210 A. Cockburn et al. / Toxicology and Applied Pharmacology 270 (2013) 209–217

Occurrence of nitrite and nitrate in feed and water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

0
Exposure assessment in livestock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
0
Exposure assessment in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
0
Risk characterisation for livestock and humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
0
Livestock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
0
Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
0
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
0
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
0

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 feeding-
stuff (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 non-
ruminants (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
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) 209–217 211

Pigs Humans
- -
NO 3 /NO2 - -
NO 3 /NO 2
Low absorption Mouth: Low bacterial High absorption Mouth: Bacterial
stomach nitrite reductase stomach nitrite reductase

Entero-salivary
Entero-salivary
circulation
circulation

Salivary uptake Salivary uptake

Urinary -
Plasma NO 3 Salivary NO2- Urinary Plasma NO 3- Salivary NO2-
excretion excretion
Acidic stomach Acidic stomach

NO NO
Plasma MetHb reductase Plasma
Low MetHb reductase (Low in neonates)

Hb MetHb Hb MetHb -
- Plasma NO3
Plasma NO3
Plasma NO2- Plasma NO 2-

Urinary Urinary
excretion excretion
Toxicity Toxicity
Cows
-/ -
NO3 NO2

Low absorption
Forestomach
-
NO2
Rumen
MetHb reductase Bacterial reductase
-
Hb MetHb PlasmaNO3
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).
212 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 gastro-
intestinal 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 sub-
acute 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 consec-
utive 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 Substrate Toxicity endpoint LOAEL NOAEL

(references) mg/kg b.w. per day mg/kg b.w. per day

Cattlea Nitrate in feed LD50 MetHb 9.9 3.3

(estimated to nitrite) Bradley et al. (1940)
Calves Nitrite per se LOAEL 34 11
Baranova et al. (2000)
Sheep Nitrite per se NOAEL 10
Trif et al. (1993)
Horsesa Nitrate in feed (estimated to nitrite) LD50 MetHb 10 3.3
Bradley et al. (1940).
Growing Pigs Nitrite in feed LOAEL 10 3.3
Koch et al. (1963)
Seerley et al. (1965)
Sows Nitrite per se Lack of developmental defects NOEL – 17.2
Sleight et al. (1972)
Rabbits Nitrite per se Urinary hormone excretion changes 13.4 4.5
Violante et al. (1973)
Poultry Nitrite per se Liver and kidney function 75 25
Sell and Roberts (1963)
Catsb Nitrite in food MetHb 69 23
FAO/WHO, 1974
Dogs Nitrite per se MetHb 7.9 2.6
Michalski (1963)
c
Fish (trout) Nitrite per se Met Hb – 0.1

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.
 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 establish-
ment 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 Com-
mittee 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 2-
year 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 sub-
chronic 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.
214 A. Cockburn et al. / Toxicology and Applied Pharmacology 270 (2013) 209–217

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

Feed commodity Country ML Mean Median Maximum Samples

group (mg/kg) (mg/kg) (mg/kg)
Nitrite (mg/kg) No N LODs or LOQs NMLs
a
Complementary feed Slovenia – 0.7 0.7 0.7 3 0 –
Complete feed Slovenia 10 2.5 1.6 7.9 15 4 0
Fish — complete feed Cyprus – 0.3 0.3 0.3 13 0 –
Fishmeal Cyprus 40 1.6 0.3 11.3 17 4 0
France 40 1.4 1.0 11.2 27 8 0
Slovenia 40 2.5 2.5 2.5 3 0 0
Forage Slovenia – 6.7 3.5 26.2 7 4 –
Other feed Slovenia – 2.6 1.7 6.5 9 3 –
Total 94 23 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 feeding-
stuffs 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 quantifica-
tion (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 concen-
tration (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 Live Consumption Nitrite intake from

weight
Total complete feed Water Total complete feed1 Water2

kg kg/day l/day mg/kg b.w. per day

Pigs 100 3.7 10 0.37 0.05

Sows 250 6.5 25 0.26 0.05
Poultry (broilers) 2.1 0.15 0.02 0.71 0.00
Poultry (laying hens) 1.9 0.115 0.02 0.61 0.001
Fish 4.5 0.09 30 0.20 3.33
1
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.
 A. Cockburn et al. / Toxicology and Applied Pharmacology 270 (2013) 209–217
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 Live weight Consumption
1 2
Forages Compound feed
Kg kg/day kg/day
Dairy cow 625 14.0 10.0.
Suckler cow 550 11.0 5.0.
Cattle 300 7.0 1.0
Lactating ewe 70 1.5 0.3
Growing lamb 20 0.45 0.15
Dairy goats 65 1.50 0.7
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, biocon-
version 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.
215
Nitrite intake from
Water Forages3 Compound feed4 Water5
l/day mg/kg b.w. per day
120 0.59 0.18 0.10
60 0.52 0.10 0.05
30 0.61 0.04 0.05
15 0.56 0.05 0.11
5 0.59 0.09 0.13
15 0.60 0.12 0.12
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). Plant-
based 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
216 A. Cockburn et al. / Toxicology and Applied Pharmacology 270 (2013) 209–217
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 inter-
conversion 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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