Food Safety

Clinic Rev Allerg Immunol (2010) 39:95–141
DOI 10.1007/s12016-009-8176-4
Food Safety
Andrea Borchers & Suzanne S. Teuber & Carl L. Keen &
M. Eric Gershwin
Published online: 13 November 2009

# Humana Press Inc. 2009
Abstract Food can never be entirely safe. Food safety is the organophosphates have been the focus of much
threatened by numerous pathogens that cause a variety of regulatory attention because there is growing evidence that
foodborne diseases, algal toxins that cause mostly acute they, too, affect the developing brain. Numerous chemical
disease, and fungal toxins that may be acutely toxic but contaminants are formed during the processing and cooking
may also have chronic sequelae, such as teratogenic, of foods. Many of them are known or suspected carcino-
immunotoxic, nephrotoxic, and estrogenic effects. Perhaps gens. Other food contaminants leach from the packaging or
more worrisome, the industrial activities of the last century storage containers. Examples that have garnered increasing
and more have resulted in massive increases in our attention in recent years are phthalates, which have been
exposure to toxic metals such as lead, cadmium, mercury, shown to induce malformations in the male reproductive
and arsenic, which now are present in the entire food chain system in laboratory animals, and bisphenol A, which
and exhibit various toxicities. Industrial processes also negatively affects the development of the central nervous
released chemicals that, although banned a long time ago, system and the male reproductive organs. Genetically
persist in the environment and contaminate our food. These modified foods present new challenges to regulatory
include organochlorine compounds, such as 1,1,1-trichloro- agencies around the world because consumer fears that
2,2-bis(p-chlorophenyl)ethane (dichlorodiphenyl dichloroe- the possible health risks of these foods have not been
thene) (DDT), other pesticides, dioxins, and dioxin-like allayed. An emerging threat to food safety possibly comes
compounds. DDT and its breakdown product dichloro- from the increasing use of nanomaterials, which are already
phenyl dichloroethylene affect the developing male and used in packaging materials, even though their toxicity
female reproductive organs. In addition, there is increasing remains largely unexplored. Numerous scientific groups
evidence that they exhibit neurodevelopmental toxicities in have underscored the importance of addressing this issue
human infants and children. They share this characteristic and developing the necessary tools for doing so. Govern-
with the dioxins and dioxin-like compounds. Other food mental agencies such as the US Food and Drug Adminis-
contaminants can arise from the treatment of animals with tration and other agencies in the USA and their counterparts
veterinary drugs or the spraying of food crops, which may in other nations have the increasingly difficult task of
leave residues. Among the pesticides applied to food crops, monitoring the food supply for these chemicals and
determining the human health risks associated with expo-
sure to these substances. The approach taken until recently
A. Borchers : S. S. Teuber : M. E. Gershwin (*) focused on one chemical at a time and one exposure route
Division of Rheumatology, Allergy, and Clinical Immunology,
(oral, inhalational, dermal) at a time. It is increasingly
University of California at Davis School of Medicine,
451 Health Sciences Drive, Suite 6510, recognized, however, that many of the numerous chemicals
Davis, CA 95616, USA we are exposed to everyday are ubiquitous, resulting in
e-mail: [email protected] exposure from food, water, air, dust, and soil. In addition,
many of these chemicals act on the same target tissue by
C. L. Keen
Department of Nutrition, University of California at Davis, similar mechanisms. “Mixture toxicology” is a rapidly
Davis, CA 95616, USA growing science that addresses the complex interactions
96 Clinic Rev Allerg Immunol (2010) 39:95–141
between chemicals and investigates the effects of cumula- TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin

tive exposure to such “common mechanism groups” of TDI Tolerable daily intake
chemicals. It is to be hoped that this results in a deeper TWI Tolerable weekly intake
understanding of the risks we face from multiple concurrent vCJD Variant Creutzfeldt-Jakob disease
exposures and makes our food supply safer. USEPA US Environmental Protection Agency
USFDA US Food and Drug Administration
Keywords Infection . Food allergies . Food additives . USDA US Department of Agriculture
Toxicology . Diarrhea . Food safety ZEA Zearalenone (a mycotoxin)
Abbreviations
ACh Acetyl choline
AChE Acetyl cholinesterase Introduction
ADI Acceptable daily intake
AGD Anogenital distance There can never be an absolute guarantee that our food is
AR Androgen receptor safe. It is simply impossible to test every single item for
ATSDR Agency for Toxic Substances and Disease every imaginable toxin, contaminant, adulterant, or food-
Registry borne pathogen, not to mention that this would make our
BBP Benzyl butyl phthalate food prohibitively expensive. Every country has an agency
BSE Bovine spongiform encephalopathy that oversees food safety, defined as a “reasonable certainty
bw Body weight of no harm,” and regulates what additives are allowed in
CDC Centers for Disease Control and Prevention food and what levels of unavoidable contaminants are
CONTAM Panel on Contaminants in the Food Chain acceptable. In the USA, the Food and Drug Agency
(EU) (USFDA) is responsible for the safety of all foods except
DAP Dialkyl phosphate meat, poultry, and egg products, which are regulated by the
DBP Di(n-butyl) phthalate Food Safety Inspection Service (FSIS) of the US Depart-
DDT 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane ment of Agriculture (USDA). In addition, the Environmen-
(dichlorodiphenyl dichloroethene) tal Protection Agency (USEPA) regulates drinking water
DEHP Di-(2-ethylhexyl) phthalate from public systems and pesticides. In order to determine
DEP Diethyl phthalate acceptable levels of contaminants and toxins, the responsi-
EFSA European Food Safety Authority ble agencies regularly monitor the food supply, and if their
EU European Union own research or scientific discoveries indicate a new hazard
DON Deoxynivalenol (a mycotoxin) or higher risk than previously recognized from a known
FB1 Fumonisin B1 hazard, they conduct risk assessments. Risk is a function of
FSIS Food Safety Inspection Service exposure and hazard or toxicity. Therefore, risk assessment
GM Genetically modified consists of hazard identification and characterization,
IARC International Agency for Research on Cancer exposure assessments, and subsequent risk characterization.
JECFA Joint (WHO/FAO) Expert Committee for The assessment of exposure to food toxicants or
Food Additives and Contaminants contaminants requires data on the dietary intake of food
MRL Maximum residue limit items or groups that are known or are most likely to contain
NHANES National Health and Nutrition Examination the chemical of interest. There are three basic approaches to
Survey determining dietary intake: (1) total diet study, (2) survey of
NOAEL No observed adverse effect level individual households or individuals, using prospective
NRC National Research Council food records or dietary recall, and (3) duplicate diet studies.
OP Organophosphate Data on dietary intake then need to be combined with
OTA Ochratoxin A databases (e.g., from governmental monitoring programs)
OVA Ovalbumin on the concentration of the contaminant of interest in foods.
PCB Polychlorinated biphenyl One of the challenges facing risk assessors is that food
PCDD Polychlorinated dibenzo-p-dioxin consumption databases were generally compiled by nutri-
PCDF Polychlorinated dibenzofuran tionists, who were interested in assessing nutrient intake.
PMTDI Provisional maximum tolerable daily intake Such databases do not necessarily contain detailed data on
PTWI Provisional tolerable weekly intake the food groups most likely to contain the additive or
Rfd Reference dose (set by the USEPA) contaminant of interest. Therefore, these databases need to
SCF Scientific Committee for Food be adjusted, or new surveys need to be conducted.
Clinic Rev Allerg Immunol (2010) 39:95–141 97
The approaches currently in use for combining dietary relationship to possible human health risks is also highly
intake and contaminant concentration data in order to arrive desirable.
at a dietary exposure estimate are either deterministic or Once dietary exposure data are available, the next step is
probabilistic. In the deterministic approach, dietary expo- to determine whether this level of exposure constitutes a
sure is calculated by multiplying a fixed value for human health risk. For many food toxicants and contam-
consumption of a food (usually the mean population value) inants, data on their toxicities are only available from
with a fixed value for the chemical concentration in that studies in laboratory animals, most commonly rodents. For
food (usually the mean concentration or the maximum level regulatory purposes, governments distinguish between
permitted). Then, the intake from all foods is summed in genotoxic substances and nongenotoxic substances (includ-
order to arrive at a point estimate. This is a relatively ing those that are carcinogens by nongenotoxic mecha-
simple, straightforward approach, yielding results that can nisms). For nongenotoxic substances, the most sensitive
be easily understood, but it has the major drawback of not toxicity end point is established from the available data, and
providing insight into the range of possible exposures and the no observed adverse effect level (NOAEL), i.e., the
the proportion of the population that remains at risk. dose at which no detrimental effects are seen in laboratory
Semiprobabilistic or simple distribution models use a fixed animals, is determined. It needs to be taken into account
value for the concentration of the chemical of interest in that there are interspecies differences and that humans may
food but employ simple distributions of food intake. In exhibit substantial differences in their sensitivity to certain
many cases, this still yields data only on the upper-bound insults due to differences in metabolic pathways and other
estimate of exposure. The probabilistic approach takes into factors. Therefore, in extrapolating from toxicities observed
account the variability in food consumption and consumer in laboratory animals to health risks in humans, uncertainty
body weight (bw) as well as the variability in contaminant factors are applied, most commonly a factor of 10 for
concentrations by using data on the total distributions of interspecies differences and a factor of up to 10 (depending
consumption as well as contaminant/toxin content of foods. on the extent and quality of the available human data) for
This is particularly important for many substances, such as human variability. According to the definition provided by
veterinary drug residues in meat or pesticide residues on the Joint (World Health Organization (WHO)/Food and
fruits and vegetables, which cannot be detected in a Agriculture Organization (FAO)) Expert Committee for
majority of samples. By representing each uncertain Food Additives and Contaminants (JECFA), the resulting
variable as a distribution function rather than a single tolerable daily intake (TDI) values “provide an estimate of
value, this method can be used to determine the likelihood the amount of a substance in food or drinking water,
with which a certain exposure level will occur. Which expressed on a body weight basis, that can be ingested daily
model is most appropriate appears to critically depend on over a lifetime without appreciable risk (standard human=
the distribution of the data on occurrence in food, e.g., 60 kg).” These intake values are referred to as acceptable
undetectable levels in many foods and low levels in much daily intake (ADI) by the USFDA, whereas the USEPA
of the remainder or low levels in many foods and very high, uses the term reference dose (Rfd). Even though regulatory
but variable, concentrations in a significant number of agencies (or the expert committees or panels advising them)
samples. generally rely on the same data, they frequently reach quite
In some cases, measuring contaminant levels in food different conclusions concerning the level of human
may not be feasible (e.g., because of laboratory contami- exposure they deem acceptable. These differences arise
nation with the chemical to be measured, as in the case of when the experts judge differently on the quality of the
phthalates). In other cases, dietary exposure is not the only existing studies and on their relevance to humans.
or not even the major route of exposure, and measurements The TDI or ADI is then used to determine the
in other media (air, dust, soil, water) may be difficult. For maximum allowable levels of a particular chemical in a
the purposes of total exposure measurements, it is therefore specific food, depending on the extent to which this food
desirable to conduct biomonitoring studies. The most contributes to the overall intake of that chemical. These
commonly used biomarkers of exposure are the concen- are called maximum limits for some chemicals and
trations of the parent compound or its metabolites in urine maximum residue limits (MRLs) for substances such as
or, more rarely, in plasma or serum. In order to extrapolate pesticide and veterinary drug and hormone residues, the
to the level of exposure that results in these biomarker latter being referred to as tolerances rather than MRLs in
concentrations, it is necessary to have information on the the US.
extent of absorption of the parent compound, its metabo- For genotoxic carcinogens, there is no dose without
lism, and the relative abundance of the resulting metabolites adverse effects, and regulatory agencies apply the “accept-
and, for urinary measurements, their fractional excretion. able risk concept.” The approach is to determine the
Knowledge on the toxicity of the metabolites and their additional cancer risk from lifetime exposure to low doses
of the chemical. What level of excess cancer risk is deemed thought to be the single most common cause of gastroen-
acceptable is the result of social convention, but frequently teritis in people of all age groups [1].
this is a value of one additional case per million. In the case One of the objectives of the US Healthy People 2010
of genotoxic carcinogens, the aim is to keep the exposure initiative is to reduce infections caused by foodborne
level as low as technologically achievable. pathogens and another is to reduce outbreaks of infections
Regulatory agencies around the world most commonly caused by key foodborne pathogens. It is well recognized
take a chemical-by-chemical approach to risk assessment. that prevention constitutes the most important measure for
However, it is increasingly acknowledged that we are reducing foodborne infections. For this purpose, The
exposed to hundreds of chemicals on a regular basis and USFDA regularly conducts food field exams, inspections,
that many of these chemicals may share a common mode of and sample collection for further analysis. Note that
action and affect the same target organ(s) or tissue(s). The monitoring of the food supply for viruses is currently
new approaches required for the risk assessment of impossible because of the lack of a simple validated
mixtures and the data that have emerged from the fairly method. Standard methods for assessing viral inactivation
new, but rapidly expanding, field of mixture toxicology will are also unavailable since viruses frequently cannot be
be discussed at the end of this paper. propagated in cell cultures and no suitable animal models
exist. In addition, the USFDA publishes guidance on how
to prevent microbial contamination of foods and is involved
Foodborne diseases in the training and education on hygiene measures of
growers and food handlers in the entire food chain since it
Bacterial, parasitic, and viral foodborne diseases has been recognized that improper storage (e.g., at
inappropriate holding temperatures), improper preparation
According to the Centers for Disease Control and Preven- (inadequate cooking), poor personal hygiene among food
tion (CDC), foodborne diseases arising from a known handlers, and contaminated equipment are major contrib-
pathogen are responsible for an estimated 14 million utors to outbreaks of foodborne diseases. Since the vast
illnesses, 60,000 hospitalizations, and 1,800 deaths each majority of food is prepared at home, the education of
year in the USA (www.cdc.gov/ncidod/dbmd/diseaseinfo/ consumers on improving the way they store and cook
foodborneinfections_t.htm). The Foodborne Diseases Ac- food is another task.
tive Surveillance Network, a collaborative effort of the Another aspect of prevention is the targeting of
CDC, USDA, and USFDA along with selected state health educational messages to persons at higher than average
departments, conducts active surveillance for seven bacteria risk of foodborne illness from particular pathogens,
and two parasites that cause foodborne diseases in a defined specifically those with primary immune defects or second-
population of almost 46 million Americans (~15% of the ary immunodeficiency (for example, human immunodefi-
US population). Their data indicate that the 2008 incidence ciency virus, chemotherapy, or organ transplantation) and
(in cases per 100,000 people) of laboratory-confirmed pregnant women [2,3]. Pregnant women have an impaired
infections was 12.68 for Campylobacter, 16.2 for Salmo- ability to clear intracellular pathogens due to the immuno-
nella, 6.59 for Shigella, 2.25 for Cryptosporidium, 1.12 for suppressive effects of pregnancy, which evolved to main-
Escherichia coli O157, and below one for the other tain the fetus. Depending on the particular immune
pathogens included in the surveillance. phenotype, a consumer may be more susceptible to certain
The major bacterial pathogens involved in foodborne bacterial foodborne illnesses, as with L. monocytogenes in
diseases include over 2,300 types of Salmonella, over 30 pregnancy, and to chronic colonization or symptomatic
types of Shigella, Campylobacter jejuni, and strain 0157: disease with intestinal parasites that are also frequently
H7 as well as several other strains of E. coli. In addition, waterborne, such as Cryptosporidium or Giardia lamblia
Listeria monocytogenes, Clostridium botulinum, Staphylo- [4]. L. monocytogenes is an intracellular bacterium that can
coccus aureus, Vibrio, and Yersinia as well as certain have devastating effects on a fetus and infect other
parasites like Cryptosporidium, Cyclospora, and Giardia immune-compromised individuals, including the elderly.
can cause foodborne disease. See Table 1 for the transmis- If an outbreak of foodborne disease occurs, the CDC is
sion routes and the symptoms these pathogens cause. In responsible for investigating the outbreak and identifying
addition to bacteria and parasites, foodborne viruses are its cause. Once it identifies possible foods, the food product
implicated in an increasing number of disease outbreaks. implicated determines which regulatory agency has primary
They can be divided into viruses that cause gastroenteritis jurisdiction. This agency is then notified and subsequently
and enterically transmitted hepatitis viruses (e.g., hepatitis attempts to trace the outbreak back to a specific source and
A virus). Examples of viruses that cause gastrointestinal to remove this source from the market as quickly as
symptoms are norovirus and rotavirus. The former is possible.
Zoonoses from cows to humans [7]. This is thought to have occurred

via the consumption of beef carrying the infective agent.
Zoonosis is defined as an infectious disease that can be Like classic CJD, this degenerative neurological disorder is
transmitted from other animals to humans. The infectious incurable and invariably fatal, usually within a few months
agents can be parasites, fungi, bacteria, viruses, and, as to a year. Altogether, there have been about 200 cases of
most recently discovered, pria (or prions). It is thought that vCJD worldwide, 162 of them in the UK [8]. Although it
zoonoses usually result from direct contact with infected has been claimed that the human risk from BSE was
animals, but zoonotic pathogens include E. coli 0157:H7, recognized in the UK from the beginning, one wonders
Campylobacter, Salmonella, and Caliciviridae (subdivided why tissues likely to contain the highest concentrations of
into two genera, Norovirus and Sapovirus), which can also the transmissible agent (brain, spinal cord, tonsil) were not
cause foodborne disease via fecal–oral contamination. The banned for human food use until 1989. Spleen and thymus
zoonosis that has garnered by far the most attention in were added to the list in 1994. After the first cases of vCJD
recent years is the emergence of a new transmissible were recognized as probably linked to BSE, the UK
spongiform encephalopathy in humans, namely variant government restricted the use of cattle for human food to
Creutzfeldt-Jakob disease (vCJD), which is thought to have animals under the age of 30 months since BSE is rare in
been caused by the consumption of meat from cows animals that are less than 30 months old. The sale of beef
infected with bovine spongiform encephalopathy (BSE) on the bone was banned in 1997 [5]. Cases of BSE also
[5]. This disease is called a prion disease because there is occurred in other European countries, but with a much
strong evidence that it is caused by an incorrectly folded lower incidence [8]. In 1996, the European Union (EU)
isoform of prion proteins that converts native prion proteins banned the import of cattle and beef from the UK. Once the
into replicates of the infectious prion isoform. This triggers epidemic was declining in the UK, the ban was eased in
a chain reaction that results in the conversion of more and 1999 to allow export of boneless beef products from
more native prions into infectious prion isoform replicates. animals 6–30 months of age. The complete lifting of the
These then form aggregates that disrupt cell function and ban did not come until 2006. All EU countries maintain an
cause cell death. Native prion proteins are normal constit- active surveillance system for the monitoring of BSE in
uents of cell membranes in vertebrates and are found at cattle [5].
particularly high concentrations in nervous tissue, which is Many of the steps taken by the USFDA in order to
the tissue affected by BSE and vCJD. The first case of BSE protect US consumers from BSE mirror those taken by the
was diagnosed in the UK in 1986, although cases are UK government, although they generally came consider-
thought to have occurred as early as in the 1970s, and ably later. In 1997, the agency banned the use of most
several cases were retrospectively diagnosed in 1985. Since mammalian proteins in ruminant feed and started routine
then, more than 184,500 cases have been reported in the testing of cows for BSE. After the emergence of the first
UK alone, the peak incidence occurring in 1992 (close to case of BSE in the US, the USFDA elaborated an
36,700 cases) [5]. Once it was recognized that meat and emergency response plan. In 2004, it prohibited specified
bone meal used in concentrated cattle feed was the most risk materials (brain and spinal cord from cattle >30 months
likely source of infectious material, the use of ruminant of age and other materials likely to contain high levels of
protein in ruminant feed was banned in 1988. This reduced infectious agents) from use in the human food supply, and
the number of new infections but was not entirely effective in 2008 it published a new feed rule banning the use of
in terminating the epidemic, most likely because cross- specified risk materials from all animal feed. Until now,
contamination of feed occurred in feed mills. Further there have been only a few isolated cases of BSE in the US
reductions in new infections were only achieved through a and Canada; no human cases of vCJD in association with
ban on feeding of all mammalian protein to all farm animal the consumption of domestic US beef have been reported,
species in 1996 [5]. and the risks of BSE in cattle and of vCJD in humans are
There are several other transmissible spongiform ence- considered very low [9,10].
phalopathies in various animal species, such as scrapie in
sheep and transmissible mink encephalopathy and chronic Toxins—shellfish and fish poisons
wasting disease in deer and elk, but none of these forms has
ever been reported to be transmitted from animals to A variety of toxins that are produced mainly by dino-
humans [5,6]. It was not until 1995 that the first case of flagellates but also some other algae are taken up by
vCJD was diagnosed in the UK, and a comparison of the mussels, oysters, crabs, and other aquatic species and
biochemical characteristics of the prion isoform in vCJD thereby enter the human food chain. The frequency as well
patients with that of the BSE-associated isoform revealed as the geographic distribution of harmful algal blooms has
them to be the same, suggesting that BSE was transmissible been increasing worldwide, suggesting that more people
Table 1 Common pathogens in foodborne illness (Bad Bug Book, www.fda.gov/Food/FoodSafety/FoodborneIllness/FoodborneIllnessFoodbornePathogensNaturalToxins/BadBugBook/default.
100
htm)
Pathogen Occurrence Main transmission routes Symptoms Possible complications

or chronic sequelae
Campylobacter jejuni Intestines of animals Contaminated water, raw Headache, fever, and muscle pain May lead to reactive arthritis and
and birds, raw milk, milk, raw or undercooked 2–5 days after ingestion; later neurological disorders in
untreated water, meat or poultry, or shellfish severe abdominal pain, nausea, 2–10% of patients
sewage sludge and diarrhea (sometimes bloody).
May last 7–10 days
Listeria monocytogenes Intestines of humans Raw milk, cheeses (particularly Listeriosis may begin with Intrauterine and cervical infections
and animals, soil, soft-ripened ones), ice cream, influenza-like symptoms; in pregnant women may result in
silage raw meats and poultry, raw gastrointestinal symptoms (nausea, spontaneous abortion or stillbirth
and smoked fish vomiting, and diarrhea) may
occur—usually after at least
12 h—and may remain the only
symptom. In serious cases, it can
manifest as septicemia, meningitis,
or meningoencephalitis, encephalitis,
and intrauterine or cervical infections
Salmonella Intestines and feces Raw or undercooked food of Fever, headache, nausea, vomiting, In ~2% of culture-proven cases,
of animals; Salmonella animal origin, mainly meat, abdominal pain, and diarrhea reactive arthritis may occur after
enteritidis in eggs poultry, eggs, and milk appearing 9–72 h after infection ~3 weeks; also Reiter’s syndrome
lasting 1–7 days
Shigella Human intestinal tract Person-to-person by Diarrhea and dysentery (diarrhea with Reiter’s syndrome, reactive arthritis,
fecal–oral route or fecal blood and mucus in the stools), and hemolytic uremic syndrome.
contamination of food handled vomiting, abdominal cramps, rectal Some strains are associated with
by workers with poor personal pain and fever, appearing 12–50 h fatality rates of 10–15%
hygiene after infection and lasting a few
days to 2 weeks
Escherichia coli Intestines of some Contaminated water, raw milk, Diarrhea (can be bloody), abdominal Especially young children can develop
O157:H7 mammals, raw milk, raw undercooked ground meat cramps, nausea, malaise, possibly hemolytic–uremic syndrome, which
unchlorinated water products, fruits and vegetables, fever, and vomiting within 3–8 days can cause acute renal failure and
unpasteurized apple juice after infection. Recovery usually has a case fatality rate of 3–5%
within 8–10 days. E. coli O157:H7
produces toxins (verotoxins or
Shiga-like toxins)
Vibrio vulnificus Shellfish and finfish; Raw oysters, clams, crabs Gastroenteritis within 16 h after May cause primary septicemia in
also found in all US ingestion individuals with certain underlying
coastal waters chronic diseases (this form is
associated with a 50% mortality)
Yersinia enterocolitica Soil, water, often Meats, oysters, fish, raw milk Fever and abdominal pain, frequently Can cause “primary septicemia” in
isolated from pigs, also gastroenteritis with diarrhea individuals with underlying chronic
cats, dogs, birds, and/or vomiting within 24–48 h after disease (e.g., diabetes, cirrhosis,
and beavers infection; may mimic appendicitis AIDS, leukemia). This form is
associated with 50% mortality
Clinic Rev Allerg Immunol (2010) 39:95–141
will be exposed to these toxins. According to the main
Can be mistaken for appendicitis,

Associated with reactive arthritis.
symptoms they cause, these poisonings are grouped into
paralytic (PSP), diarrhetic (DSP), neurotoxic (NSP), and
amnesic shellfish poisoning (ASP). In addition, there are
resulting in unnecessary
ciguatera fish poisoning and azaspiracid shellfish poisoning
and yessotoxin and palytoxin poisonings. See Table 2 for a
appendectomies
summary of the toxins involved, their sources, mechanisms,
or action, and the acute symptoms they cause. Many of the
dinoflagellate toxins are neurotoxins that interact with
voltage-gated sodium and or calcium channels in different
ways and cause either increases or decreases in the flux of
these ions, thereby resulting in different sets of symptoms.
PSP and NSP toxins as well as ciguatoxins, azaspiracids,
weakness in the neck and arms, paralysis

blurred vision, dry mouth, and difficulty
blood pressure and heart rate. Recovery
of the respiratory muscles, and death if

yessotoxins, and palytoxin all belong to this group.
ingestion. They include severe nausea,
released by the bacterium and are that
abdominal swelling. These symptoms

in speaking and swallowing, possibly
produce and occur within 1–6 h after
headache, muscle cramping, changed
4 h to 8 days). They can progress to

usually occur within 12–36 h (range
weakness, and vertigo, followed by
vomiting, diarrhea, constipation, or

of an intoxication: marked fatigue,
Symptoms are caused by the toxin
abdominal cramps, vomiting, and
PSP The major PSP toxins belong to the saxitoxin group,

diarrhea; in more severe cases,
enterotoxins that some strains
of which at least 29 congeners are known and which are

Symptoms are caused by the
not treated with antitoxin

produced mainly by members of the Alexandrium, Gymno-
dinium, and Pyridinium genera of dinoflagellates. Symp-
toms after ingestion of PSP toxins develop rapidly (within
within 2–3 days
0.5–2 h) and include tingling sensation of the lips, mouth,

and tongue, gastrointestinal problems, numbness of the
extremities, difficulties with muscle coordination, respira-
tory distress, and paralysis. Severe cases can proceed to
respiratory arrest and cardiovascular shock, the fatality rate
being approximately 20% [11,12]. The lethal dose in
humans is between 1 and 4 mg. The European Food Safety
garlic in oil, tightly wrapped
Authority (EFSA) set an acute Rfd (ARfd) of 0.5µg/kg bw.

meat products, poultry, egg
Food handler to food (meat,
products, milk, and dairy
Improperly canned foods,
Both the USFDA and the EFSA have a maximum limit of

products, salads, bakery
80µg/100 g (or 800µg/1 kg) of PSP toxins in saxitoxin

equivalents in shellfish tissue. Ingestion of a somewhat
large 400-g portion of shellfish containing the maximum
allowable level of saxitoxin equivalents would result in an
products)
acute exposure of 320-µg toxins or 5.3µg/kg bw in a 60-kg

food
adult. Since this intake is tenfold higher than the ARfd, it

has been suggested that more appropriate limits (e.g., a
more than tenfold reduction of the current limits) should be
considered for saxitoxin equivalents in shellfish [13].
Soil, water, plants,
animal skin, hair,
and intestines of
animals and fish
NSP Karenia brevis and several other dinoflagellates (see

Human and other
in nasal passage
also Table 2) produce hemolytic and neurotoxic substances,

and throat
the latter being designated as brevetoxins. There are a total

of ten known brevetoxins, subdivided into type 1 and type
2 based on the structure of their backbones [14]. Brevetox-
ins and some of their molluskan metabolites cause the
typical symptoms of NSP, which are milder than those of
PSP and include nausea, tingling, and numbness of the lips,
Staphylococcus aureus
Clostridium botulinum
mouth, and face, paresthesia, loss of motor control, and

severe muscular pain [11,14]. The pathogenic dose for
humans is between 42 and 72 mouse units. Note that many
of the shellfish poison levels are still measured in mouse
units, which are defined as the amount of shellfish poison
required to kill a 20-g mouse within 15 min after
Table 2 Shellfish poisoning toxins: sources, vectors, and symptoms adapted from Wang et al. [11]
Type of Toxin Sources of toxin Primary Mechanism Symptoms

poisoning vector
PSP Saxitoxins, Alexandrium spp., Shellfish Voltage-gated Tingling of perioral area,

gonyautoxins Gymnodinium sodium channel 1 gastrointestinal problems,
spp., Pyridinium spp. numbness of extremities,
disturbed muscle
coordination, respiratory
distress, paralysis;
20% mortality
NSP Brevetoxins Karenia brevis, Shellfish Neurotoxin acting Nausea, numbness of
Chattonella marina, via voltage-gated perioral area, paresthesia,
Chattonella antiqua, sodium channel 5 disturbed motor control,
Fibrocapsa japonica, severe muscular pain
Heterosigma akashiwo
Ciguatera Ciguatoxins, Gambierdiscus toxicus, Coral reef fish Voltage-gated sodium >175 gastrointestinal,
fish poisoning maitotoxins G. belizeanus, channel 5, voltage-gated neurological, cardiovascular,
G. yasumotoi calcium channel and general symptoms;
can be fatal
AZP Azaspiracids Protoperidinium crassipes Shellfish Voltage-gated Nausea, vomiting, severe
calcium channel diarrhea, stomach cramps
Palytoxin Palytoxins Palythoa toxica, Shellfish Sodium–potassium Fever, ataxia, drowsiness,
Ostreopsis siamensis ATPase often fatal
Yessotoxin Yessotoxins Protoceratium reticulatum, Shellfish Possibly voltage-gated
poisoning Lingulodinium calcium/sodium channel
polyedrum,
Gonyaulax spinifera
DSP Okadaic acids Dinophysis spp. Shellfish Inhibition of phosphatases
and of protein synthesis
ASP Domoic acids Pseudo-nitzschia spp. Shellfish Activation of the kainate Vomiting, diarrhea,
glutamate receptor abdominal cramps, severe
headache, loss of short-term
memory, can be fatal
intraperitoneal injection. Brevetoxin levels in shellfish gastrointestinal illness, whereas neurological symptoms
tissue have been set at 20 mouse units/100-g shellfish may require ~4.5 mg/kg bw [15].
or, more recently, 80µg/100-g shellfish in brevetoxin
equivalents [14]. Ciguatera fish poisoning This poisoning is caused by the
consumption of contaminated coral reef fishes, such as
ASP The only known outbreak of ASP occurred in Canada barracuda, grouper, and snapper. The dinoflagellate Gam-
in 1987 [15]. The toxins responsible for ASP symptoms bierdiscus toxicus produces maitotoxins, which are bio-
were identified as domoic acid and its ten isomers, which transformed into ciguatoxins by herbivorous fishes and
are the only shellfish toxins not produced by dinoflagellates invertebrates. Ingestion of these toxins causes >170
but by diatoms of the genus Pseudo-nitzschia. These toxins gastrointestinal, neurological, cardiovascular, and general
accumulate in a wide variety of shellfish species, including symptoms, with neurological symptoms predominating in
crabs, mussels, razor clams, scallops, and cockles, and the Pacific Ocean, whereas mostly gastrointestinal distur-
much lower levels have also been detected in anchovies and bances are seen in the Caribbean. Ciguatoxins are highly
mackerel. Symptoms after ingestion of domoic-acid- toxic, with as little as 0.1µg being sufficient to cause illness
contaminated shellfish include gastrointestinal symptoms in humans [11,12].
such as vomiting, abdominal cramps, and diarrhea and
neurological symptoms such as debilitating headache and AZP The first report of an AZP incident came from the
loss of short-term memory (seen in only 25% of patients Netherlands and was associated with Irish mussels, but the
but responsible for the name of the poisons). Three of the group of toxins causing it has since been found to constitute
107 patients that fulfilled the clinical definition of the a more widespread problem in Europe [11]. These toxins are
illness died. Rough exposure estimates suggest that 1 mg produced by Protoperidinium crassipes and are derivatives
domoic acid per kilogram bw is sufficient to induce of azaspiracid, of which at least 11 have been identified.
Ingestion of contaminated shellfish results in symptoms toxins but is actually histamine poisoning due to bacterial
similar to those seen with DSP, i.e., nausea, vomiting, severe action, with contribution from other biogenic amines [18].
diarrhea, and stomach cramps. However, in mice, the effects Numerous case series document emergency department
of AZP differ markedly from those seen with DSP in that visits (sometimes considered acute allergic reactions) [19]
they include severe neurological symptoms, such as respira- that on investigation were found to be from ingestion of
tory distress, spasms, and paralysis of the limbs. spoiled fish. Symptoms usually start within 15 min to 2 h
and can include flushing, hypotension, palpitations, loss of
DSP DSP is caused by okadaic acid and some of its consciousness, headache, skin rashes, nausea, diarrhea,
congeners, of which at least seven have been identified and vomiting, and shortness of breath or wheezing. Dark-
which are called dinophysistoxins [11]. The major pro- fleshed fish of the Scombridae family, especially, contain
ducers of this group of toxins are various Dinophysis higher levels of free histidine that is decarboxylated to
species. Acute symptoms after ingestion of contaminated histamine by bacteria. This can occur after just a few hours
shellfish include diarrhea, nausea, vomiting, and abdominal at ambient temperatures and has even been reported in fish
pain. No fatalities have been reported to date. Okadaic acid that is chilled but not adequately so. Histamine is heat
inhibits protein synthesis and also is an inhibitor of stable, so cooking the fish will not prevent the toxicity.
phosphatases. In addition, it increases DNA methylation, Persons may differ substantially in their sensitivity to the
which is an important mechanism of gene regulation. It is ingested histamine, which may be directly due to their
thought that this ability to interfere with gene regulation is endogenous diamine oxidase activity in the small intestine.
involved in the potent tumor-promoting effects that okadaic
acid exerts in laboratory animals [16]. The CONTAM panel
set a new lower ARfd of 0.3µg okadaic acid equivalents Mycotoxins
per kilogram bw [17]. As in the case of saxitoxins, a large
portion (400 g) of shellfish contaminated with the maxi- Mycotoxins are secondary metabolites produced by fungi
mum of 160µg okadaic acid equivalents per kilogram that infect a variety of crops, including cereals, nuts, spices,
shellfish would exceed the ARfd by a factor of 3. Hence, a and in some cases fruit.
reduction in the maximum level was deemed desirable [17].
Aflatoxins Aflatoxins are produced by three species of
Other shellfish poisonings Several algae toxins were Aspergillus, namely Aspergillus flavus, Aspergillus para-
originally classified as DSP because they frequently co- siticus, and Aspergillus nomius, with A. flavus synthesizing
occur with DSP toxins and are sometimes produced by the only B aflatoxins, whereas both B and G aflatoxins occur in
same species of algae. However, they were subsequently the other species [20]. Peanuts and maize are the most
discovered to not (or only weakly) cause diarrhea or inhibit frequently contaminated foods, but pistachios and other nuts
phosphatases. These include the pectenotoxins, which are can also contain very high levels. Hydroxylation of B1 and B2
produced mainly by several Dinophysis species and are aflatoxins yields M1 and M2 aflatoxins, respectively. These
hepatotoxic, and the yessotoxins, which are synthesized by metabolites are found in milk from animals that consumed
Protoceratium reticulatum and Lingulodinium polyedrum aflatoxin-contaminated feed. Aflatoxin intake is very difficult
and mainly target the heart, at least in mice [11]. to estimate because the contamination levels in foods are
Note that some of the so-called shellfish poisons are not frequently below the limit of detection. Assuming that samples
restricted to shellfish but may also occur in other commonly without detectable aflatoxins B1, B2, G1, and G2 contained
consumed fish species, though at much lower levels. In concentrations of one half the limit of quantitation, mean
addition, it is only during certain times of the year that they dietary intake estimates of 0.12 and 0.32 ng/kg bw in adults
accumulate in shellfish to levels that cause acute toxicity, and children, respectively, were obtained in a recent French
but they can be present at lower levels throughout much of total diet study [21]. Aflatoxin M1 intake was estimated at
the remainder of the year [11,12]. For example, K. brevis 0.09 ng/kg bw per day in adults and 0.22 in children (see
counts of 1,000 cells per liter of seawater are considered also Table 3). According to an investigation using a dietary
background levels at the Florida coast, and Florida shellfish questionnaire for assessing food consumption, Swedish
beds are closed only when counts are equal to 5,000 per liter adults are exposed to a mean of 0.76 ng/kg bw per day
seawater or higher [14]. Very little is known about the chronic (P95 2.1 ng/kg bw per day) [22]. In this study, a majority of
toxicity of low levels of exposure to these toxins. This is samples were above the detection limit. The rather high
particularly worrisome given that some of them are already intake was driven almost exclusively by extremely high
suspected of having carcinogenic or hepatotoxic effects. levels of contamination in Brazil nuts and pistachios.
Scombroid fish poisoning is a very common cause of Aflatoxin B1 is highly mutagenic and carcinogenic and
adverse reactions to fish that is not due to zooplankton is one of the most potent liver carcinogens known.
18.0 (56.7)
29.6 (106)
Aflatoxin M1 is approximately tenfold less potent. The
Patulin JECFA did not set a TDI for aflatoxin B1 because even
very low levels of exposure increase the risk of liver cancer.
The aflatoxin levels in food were all below the limit of quantitation. This estimate is based on the assumption that nondetectable levels are equal to one half the limit of quantitation
Instead, it was calculated that intake of as little as 1 ng/kg
bw per day would result in one extra cancer case in 105
Zearalenone
individuals. Subjects with hepatitis B infection are at

66 (132)
33 (70)
increased risk of hepatocellular carcinoma from aflatoxin

exposure. It is recommended to keep the contamination
levels as low as possible through good manufacturing and
storing practices. If contamination is present, there are a
Fumonisins
46 (175)
variety of chemical or physical means for reducing the

14 (64)
aflatoxin content [20,23].

219
355
129
Ochratoxins Ochratoxins are a group of structurally related
2.16 (3.63)
4.07 (7.77)
secondary metabolites that are produced mainly by Peni-

cillium verrucosum and Aspergillus ochraceus, occasional-
Table 3 Mean daily dietary mycotoxin intake in nanogram per kilogram body weight per day (P95 where availablea) [20–22,26,27,32]
OTA
2.31
3.39
1.71
0.53
1.42
1.09
ly also by isolates of Aspergillus niger [20]. The main and

most toxic mycotoxin in this group is ochratoxin A (OTA),
which is found in cereals, oil seeds, coffee beans, pulses,
(156)
(207)
1.2 (1.9)
1.4 (2.6)
(57)
(67)
wine, and poultry meat. In the dietary exposure assessment

performed by SCOOP, a scientific cooperation of EU states
T2
45
67
30
34
14
11
6
and Norway, the main dietary source was cereal grains in

Omitted values are either not available or are based on analysis of a very limited number of sampled food groups
most European countries, but coffee and wine made the

(143)
72 (130)
(98)
(59)
(69)
39 (69)
major contribution to exposure in Greece and Italy,

HT-2
respectively [24,25]. Dietary intake was estimated to be

30
44
26
30
12
18
6
~1–3 ng/kg bw per day (see also Table 3), but this is thought
to underestimate actual intake because not all sources were
163 (300)
88 (157)
58 (199)
94 (307)
57 (110)
taken into account in determining exposure. In a French

50 (93)
6 (13)
NIV
study on dietary mycotoxin exposure, bread was the major

17
25
64
source of OTA, accounting for one third of total intake [21].

Much of the remainder of the intake came from other flour-
(1,667)
(2,430)
containing food types. A duplicate diet study of 123 Dutch

(571)
(929)
(530)
(628)
(155)
participants indicated a mean OTA intake of 1.2 ng/kg bw

DON
78
281
451
461
725
300
343
142
176
483
per day [26], whereas in a UK duplicate diet study, where

each participant collected duplicates for 30 days and one
intake value was determined for the entire month, a mean
0.117 (0.345)
0.323 (0.888)
dietary exposure of 0.94 ng/kg bw was calculated [27].

0.76 (2.1)
Aflatoxinb
The absorption of OTA occurs in the upper gastrointes-

tinal tract and ranges between 40% and 66% in various
animal species [28]. Essentially, all OTA in blood is bound
to proteins. It is distributed mainly to the kidney and also to
Children (1.5–4.5)
liver, muscle, and fat. It has been shown to cross the

Children (7–14)
placenta in different animal species and has been detected

Adult females
Female adult
Adult males
Male adult
in human breast milk [29,30]. There are substantial

Children
Children
interspecies differences in serum half-lives, ranging from

Adults
Adults
18–74
Adult
1 day in mice to 21 days in monkeys and ~35 days in

humans. Excretion occurs via bile and urine, and there are
P95: 95th percentile
indications of enterohepatic circulation.

Norway (SCOOP)
Sweden (SCOOP)
Finland (SCOOP)
France (SCOOP)
OTA has been shown to be nephrotoxic in almost all

UK (SCOOP)
animals species investigated to date. In humans, OTA

exposure is thought to be associated with Balkan endemic
Germany
Sweden
France
France
nephropathy, but a causal connection has not been proven

so far [31]. At much higher doses than those needed to
b
a
induce progressive nephropathy in animals, OTA has DON, the SCF concluded that the limited data available did
hepatotoxic, teratogenic, and immunotoxic effects. In not support the establishment of a group TDI, and they set a
addition, it has been reported to cause kidney tumors in final TDI of 1µg/kg bw. At the high end of DON intake,
mice and rats, with male animals being more sensitive than mean dietary exposures far exceeded the TDI in some
females. The International Agency for Research on Cancer European countries [34].
(IARC) classified it as a probable carcinogen to humans DON is rapidly and extensively absorbed in swine,
(group 2B) [31]. The JECFA set a provisional maximum TDI followed by wide but transient tissue distribution and rapid
of 0.014µg/kg bw (see also Table 4) [23]. excretion, the elimination half-life being only 3.9 h. Studies
in rodents also indicate that DON does not accumulate in
Fusarium toxins A variety of Fusarium species which can the body. Ruminants and poultry show very limited
infect cereal crops in the field synthesize toxins. Toxin absorption and little susceptibility to the toxicity of this
production occurs mainly before harvesting but can take compound [36]. In various animal species, a major effect of
place postharvest, if the crop is not handled properly. There subchronic/chronic exposure to DON is decreased weight
are three major groups of Fusarium toxins, the trichothe- gain and anorexia due to feed aversion. This is also thought
cenes, fumonisins, and zearalenone (ZEA). to be responsible for the fetal toxicity and teratogenicity,
Trichothecenes are structurally related sesquiterpenoids which are generally observed only at levels that induce
produced by several fungi and are subdivided into four maternal toxicity. In several animal species, DON is
categories depending on their functional groups. Major associated with immunotoxicity, including impaired
representatives of the type B trichothecenes are deoxyniva- delayed type hypersensitivity responses, antigen-specific
lenol, nivalenol, and 3-acetyldeoxynivalenol, while T-2 antibody production, and host resistance and altered
toxin and HT-2 toxin are type A trichothecenes. cytokine production. In rodents, but not in swine, serum
Estimates of mean dietary intake of deoxynivalenol total IgA (and sometimes IgG) levels are markedly
(DON) in various European countries ranged between 78 elevated, resulting in IgA immune complexes that are
and 725 ng/kg bw per day [32–34]. The major sources were deposited in the kidneys, resulting in a glomerulonephritis
cereals and cereal products, particularly corn [35]. The that resembles human IgA nephropathy [36]. Whether DON
JECFA, the Scientific Committee for Food (SCF) for the exerts similar effects in humans remains to be investigated.
EU, and the Nordic Working Group have all conducted risk T-2 and HT-2 are produced mainly by Fusarium
assessments and established TDIs for some of the tricho- sporotrichioides and to a lesser extent by Fusarium poae,
thecenes (see also Table 4): some of these values are Fusarium equiseti, and Fusarium acuminatum, affecting
provisional or temporary because Fusarium species are mostly corn, wheat, and oats. In a recent European survey,
capable of producing several trichothecenes, and these may dietary intake of T-2 and HT-2 was found to exceed the
share a common mechanism of toxicity, making it desirable European group TDI of 0.06µg/kg bw per day in a large
to take cumulative effects into account 31. In the case of proportion of the population, with some infants reaching
Table 4 TDI values of mycotoxins in microgram per kilogram body weight per day [23,32,33]
Type of toxin Specific toxin SCF/EU JECFA Nordic USEPA (Rfd) Canada
working group
Type B Deoxynivalenol 1.0 1.0a 1.0b

trichothecenes
Nivalenol 0.7b Insufficient data
Type A T-2 toxin 0.06b 0.6a 0.2b
trichothecenes HT-2
Zearalenone 0.2b 0.07a, c
0.1b 0.1b
Fumonisins Fumonisin B1 2.0 2.0a
Fumonisin B2
Fumonisin B3
Ochratoxins Ochratoxin A 0.005 0.014c 0.005 0.12 0.0012–0.0057
Patulin Endorsed the 0.4a
JECFA value
a
Provisional maximum tolerable daily intake
b
Temporary TDI
c
Calculated from a provisional tolerable weekly intake
>500% of the TDI (see Tables 3 and 4) [32]. Note, however, Fumonisins are produced by certain Fusarium species
that <20% of the samples tested positive, so the question of that mainly contaminate maize and maize products but also
how samples below the limit of detection are treated other cereal grains [20]. Accordingly, the major dietary
(considered to be zero or equal to one half of the limit of sources of fumonisins in various European countries were
quantitation) has a major influence on the estimate. Wheat cereals, particularly corn and wheat products. The most
and products made from wheat (e.g., bread and pasta) toxic compound is fumonisin B1 (FB1), and this is also the
constituted the predominant dietary source of these toxins. fumonisin for which there are the most data. See Table 3 for
T-2 is rapidly metabolized to HT-2, making it difficult to mean dietary intake levels. FB1 is poorly absorbed and
determine which of the two compounds is responsible for the rapidly eliminated; however, there are indications that a
toxic effects. Acute poisoning is associated with nausea, small pool of FB1 or its metabolites are retained in the liver
vomiting, abdominal pain, diarrhea (at times bloody) and kidneys. It is therefore not surprising that FB1 targets
dizziness, and chills and is lethal in a high proportion of the liver and kidney in almost all species examined today,
cases. The chronic toxicity is a result of the potent ability of with mice and rats showing the greatest susceptibility. In
T-2 (and HT-2) to inhibit protein synthesis, which is most addition, FB1 has species-dependent organ-specific toxic-
obvious in the immune system. Decreased antibody produc- ities, such as porcine pulmonary edema in pigs and equine
tion and delayed type hypersensitivity responses as well as leukoencephalomalacia in horses.
changes in leukocyte counts due to selective depletion of In mouse embryo cultures and in LM/Bc mice, FB1
leukocyte precursors have been observed. In addition, there causes neural tube and craniofacial defects, which can be
is concern that T-2 can have carcinogenic effects [20]. partially prevented by supplementation with folic acid,
ZEA is another mycotoxin produced by several fungi of while ganglioside supplementation is almost completely
the genus Fusarium [33]. It occurs mostly in maize (corn) protective [37]. The mechanism is thought to involve the
but can contaminate other cereal grains and also soybeans. ability of fumonisins to disrupt sphingolipid biosynthesis. The
ZEA usually is present together with other Fusarium resulting lack of complex sphingolipids impedes the function
toxins, such as trichothecenes and fumonisins. Whereas of certain membrane proteins, including the folate-binding
the main sources of ZEA intake in Northern America are protein. Further support for the hypothesis that fumonisin
corn and wheat products, in Europe they are wheat, rye, and exposure is causally involved in neural tube defects comes
oats. Intake estimates are 20 ng/kg bw per day for Canada, from a study of Mexican–American women living along the
Denmark, and Norway and 30 ng/kg bw per day in the Texas–Mexico border, an area with a high incidence of these
USA (see also Table 3). Small children may have intakes as birth defects [38]. The results of this investigation indicated
high as 55 ng/kg bw per day [33]. This is well below the that maternal corn tortilla intake (as a surrogate for fumonisin
tolerable intake values established by various regulatory exposure) during the first trimester of pregnancy and a
agencies (between 0.1 and 0.5µg/kg bw per day, see also biomarker of fumonisin exposure (the sphinganine-to-
Table 4). After oral administration, ZEA is readily absorbed sphingosine ratio in postpartum serum) were significantly
in rats, rabbits, pigs, and humans. It is then extensively associated with the occurrence of neural tube defects.
metabolized in the intestine and/or liver, with hydroxylation Furthermore, there is an elevated incidence of neural tube
resulting in the formation of α- and β-ZEA. Intestinal cells defects in other areas where high dietary intake of fumonisins
also seem to be able to form α- and β-zearalenol. ZEA and is likely (Guatemala, South Africa, and China). Of note, it has
its metabolites are conjugated with glucuronic acid, which been generally assumed that FB1 does not cross the placenta.
would facilitate their urinary excretion. There are indica- However, disturbances in fetal sphingolipid profiles and
tions of extensive enterohepatic recirculation and, conse- folate receptor expression after maternal FB1 supplementa-
quently, elimination via feces. Whether and to what extent tion, as observed in LM/Bc mice [37], suggest that FB1 or its
these processes occur in humans remain to be elucidated metabolites can be transferred across the placenta.
[20]. ZEA has fairly low acute toxicity. In chronic exposure
studies, it exhibits hematoxicity and hepatotoxicity in rats Patulin Patulin is a secondary metabolite produced by
and rabbits, and high doses are associated with hepatocel- fungi belonging to several genera, including Penicillium,
lular and pituitary adenomas [20]. At much lower doses Aspergillus, and Byssochlamys. Although many fruits,
than are required for carcinogenic effects, ZEA shows grains, and other foods can be infected, the commodities
estrogenic activity, resulting in reproductive toxicities in with the highest concentrations are generally apples and
various animal species, with pigs and sheep being more apple juice, and these are also the major sources of dietary
sensitive than rodents. These include decreased fertility, intake [39]. Mean intakes for the populations of various EU
reduced litter size, altered weight of adrenal, thyroid, and countries and Norway generally ranged between 1.1 and
pituitary glands [33]. The IARC considered it as not 9.6 ng/kg bw per day, with children having several-fold
classifiable as to its human carcinogenicity (group 3). higher mean dietary exposures. Available data indicate that
patulin has no reproductive or teratogenic effects and manifests as peripheral neuropathy involving extensor
exhibits embryotoxicity only at doses that are toxic to the muscles, but not sensory nerves. The developing brain is
mother. It is not mutagenic but induces chromosomal much more susceptible to the neurotoxic effects of Pb than
damage. A provisional maximum tolerable daily intake of the adult brain. Numerous studies have shown that Pb
0.4µg/kg bw was established by JECFA [40]. exposure in infants and children is associated with learning
disabilities, decreased IQ, behavioral problems, and dis-
turbances in fine motor function. Some of these effects are
Substances entering the food chain seen below blood Pb concentrations of 10µl/dl, which the
from the environment CDC and the American Pediatric Association consider the
level of concern. Of note, among the children examined as
Heavy metals part of the nationally representative National Health and
Nutrition Examination Survey (NHANES) III (1999–2000),
Lead (Pb) Since the phasing out of leaded gasoline, major the upper bound of the 95% confidence interval of the 95th
sources of high Pb exposure are Pb paint (still found in an percentile of blood Pb concentration was 9.90 in the 1–5-
estimated 38 million homes in the USA) and drinking water year-old age group [43].
contaminated from Pb pipes or brass fixtures, which may Another Pb-associated toxicity is kidney damage, which
contain up to 8% of this metal. However, lead also persists in is not only well established in occupational Pb exposure but
the environment, including soils that food crops are grown is seen at much lower exposures in the general population.
on, and food appears to be the major source of Pb exposure. In addition, Pb exposure is associated with an increased risk
Local Pb contamination of pastures can result in consider- of hypertension, cerebrovascular, and cardiovascular dis-
able contamination of meat and milk, and fish generally also ease. Furthermore, the IARC has reclassified Pb from a
contain high Pb concentrations. In some recent analyses possible to a probable human carcinogen, since there is
from Spain, the food groups “fish and shellfish” and “cold increasing evidence of an association between Pb exposure
meats and sausages” were found to contain the highest Pb and overall cancer incidence but particularly stomach, lung,
concentrations, but substantial amounts were also detected and bladder cancer. WHO set a provisional tolerable weekly
not only in meat, eggs, and dairy products but also in fruits, intake (PTWI) of 25µg/kg bw. In contrast, the USEPA and
vegetables, cereals, and tubers and in alcoholic beverages Agency for Toxic Substances and Disease Registry
[41,42]. The highest contribution to adult Pb intake came (ATSDR/CDC) agree that there is no threshold for lead
from fish and shellfish, whereas cereals were the major below which no harm occurs.
source of Pb for children and adolescents [41]. In Lebanon,
bread accounted for up to 28% of total dietary Pb intake. In Mercury (Hg) It is estimated that less than 50% of global
the Canary Islands, Spain, water contained only 7.3µg Pb Hg releases arise from natural sources, with the remainder
per kilogram but was estimated to constitute ~20% of daily coming from human activities, such as burning of coal,
Pb intake at an average consumption of 2 l/day [42]. Mean cement production, mining, and metal processing. Anthro-
dietary intake values are summarized in Table 5. pogenic emissions, particularly from Asia, are expected to
The extent of gastrointestinal Pb absorption depends on increase significantly in coming decades. Long-range
nutritional status, with iron deficiency resulting in increased transport results in the deposition of Hg throughout the
and calcium supplementation in decreased Pb absorption. hemisphere in which it was emitted. When inorganic Hg
Age is another factor that influences Pb absorption. reaches aquatic environments, sediment bacteria convert it
Generally, children have a much higher capacity for into methyl mercury (MeHg), which is then bioaccumulated
absorbing orally ingested Pb (up to 75%) than adults (only and biomagnified in the aquatic food chain. As a result, fish
10–15%). Once absorbed, the metal is then slowly (over a at the topmost trophic levels contain the greatest concen-
period of 4–6 weeks) distributed to various tissues, trations of MeHg, as do sea mammals. More than 90% of
including liver, renal cortex, brain, and bone, the latter total Hg in fish is thought to be present in the form of
constituting the major storage site for Pb. Pb is remobilized MeHg. The highest levels of MeHg are found in predatory
from the exchangeable pool at the bone surface, a process species such as shark, swordfish, marlin, pike, large tuna,
that is particularly obvious during conditions associated and king mackerel. As a result, humans are exposed to this
with increased bone turnover, including pregnancy and metal mainly through the consumption of fish and, if
lactation. Inorganic lead does not undergo metabolism, and applicable, sea mammals. Using fish meal as animal feed
the majority is excreted via urine, while the extent of fecal can result in substantial levels of MeHg in meat and other
excretion remains to be established. animal products. Table 5 summarizes mean dietary intake
The primary target of lead toxicity is the central and values for total Hg. The ethyl mercury compound thimer-
peripheral nervous system. In adults, this most commonly osal, which is used as a preservative in certain childhood
Table 5 Dietary intake of lead (Pb), total mercury (Hg), total arsenic (As), and cadmium (Cd) in microgram per day (from total diet studies unless
indicated otherwise)
Country Age group Lead Mercury (total) Arsenic Cadmium
US 1982–1984 Infants (6–11 months) 0.49

Children (2 years) 1.3
Female adults 2.9
Male adults 3.9
US 1986–1991 Female adults 8.2 50.6
Male adults 8.6 58.5
NHEXAS Maryland 8.14
NHEXAS Arizona (aggregate lead exposure) Male adults 42
Female adult 35
Children (<18 years) 27
Greenland 2.1 121 143
France (duplicate diet from mass catering establishments) 52 109 17
France Adults 57 8.48 148 10.6
Children (3–14) 40 5.86 101 7.8
Germany Adults 47 9.91 37 19.2
Children (4–6) 26 5.56 20 13.6
Children (10–12) 34 7.51 27 17.4
UK Adults 27 3.02 66 12.1
Denmark Adults 18 3.5 37 16
Catalonia, Spain Children 23.0 18.63 132.82 13.2
Adolescents 25.7 16.57 157.82 14.8
Female adults 23.1 16.71 190.38 12.0
Male adults 28.4 21.22 223.59 15.7
Seniors 23.4 16.95 192.12 12.7
Catalonia, Spain Adults 59.3 9.89 261.01 10.0
Canary Islands, Spain 58.2a 11.2
Beirut, Lebanon 15.5a 9.3
Japan (duplicate diet) 1980 Female adults Female adults 37.5
Male adults 52.0
Japan (duplicate diet) 1995 Female adults 26.2
Male adults 26.7
a
After subtracting 14.6µg/day for water, which was not included in the other analyses
vaccines, is another source of exposure. Dental amalgams elimination from the body. The half-life in the body is
may also make substantial contributions to total exposure. ~50 days. Of note, bacterial demethylation and biliary
MeHg after fish consumption is almost completely excretion are not observed in suckling animals, and a
absorbed and distributed throughout the body within the similar inability to metabolize MeHg may underlie the
following 30–40 h. It accumulates particularly in the liver failure of human neonates to excrete this compound.
and kidney, but about 10% is found in the brain. In addition Numerous animal studies have shown that MeHg is a
to crossing the blood brain barrier, MeHg can cross the developmental neurotoxicant and also has potent neurotoxic
placenta, resulting in similar or higher levels in cord blood effects in adult animals [44]. Even at exposure levels well
compared to maternal blood, whereas the fetal brain below those that affect the central nervous system, MeHg
exhibits at least fivefold higher concentrations than mater- can profoundly affect the immune system. The extent and
nal blood. The intestinal microflora can metabolize MeHg nature of these immunotoxicities depends on the timing,
to inorganic Hg, which is either excreted via bile or slowly dose, type of Hg compound (metallic, inorganic, or
accumulates in the body but is thought to be present in inert organic), and the genetic characteristics of the host. For
form. Urinary excretion accounts for less than 10% of total example, depending on the mouse strain, inorganic Hg was
found to reduce thymus weight, yet may either enhance, reduced its PTWI from originally 3.3 to now 1.6µg/kg bw
decrease, or not significantly affect the lymphoproliferative (corresponding to 0.23µg/kg bw per day). Note, however,
response. In addition, exposure to inorganic Hg results in that a single meal of one of the more highly contaminated
polyclonal B and T cell activation, specific nucleolar species (such as swordfish or marlin) would result in an
autoantibody production, deposition of immune complexes intake of >2µg/kg bw, which vastly exceeds the USEPA
in the kidney, and glomerulonephritis in susceptible mouse Rfd and is higher even than the PTWI of the FAO/WHO.
strains. A similar autoimmune syndrome is observed in The USFDA therefore advises pregnant or nursing women
certain rats. Clinical manifestations subside spontaneously and young children to avoid highly contaminated species
in both species but can be reinduced in mice, whereas rats and to eat fish at most twice a week.
become resistant due to the development of suppressor CD8
T cells. Like inorganic Hg, organic Hg decreases thymus Cadmium (Cd) This element occurs naturally in ore,
weight but can augment the lymphoproliferative response. usually as Cd oxide, Cd chloride, Cd sulfate, or Cd sulfide
It is also capable of inducing autoantibody production in and enters the environment from weathering of Cd-
susceptible animals but does so with much slower kinetics, containing rocks as well as mining activities. The produc-
suggesting that conversion to inorganic Hg is required. Of tion and emission of Cd has increased dramatically over the
note, occupationally exposed cohorts exhibit increased last century due to its use as an anticorrosion agent,
levels of several autoantibodies as well as T cell lympho- stabilizer in polyvinyl chloride (PVC) products, color
proliferation. This suggests that the ability of Hg to break pigment, and most commonly now in rechargeable nickel–
tolerance and induce autoimmunity may be of human Cd batteries.
relevance, at least for genetically susceptible populations. A major source of Cd exposure is cigarette smoking, but
Several large-scale poisoning incidents involving highly food constitutes the most important contributor in non-
contaminated fish in Japan in the 1950s and 1960s and seed smokers. The Cd content of crops grown for human
grain that was used for human consumption in Iraq in the consumption reflects the Cd concentration of the soil they
1970s have highlighted that the major target of MeHg were grown on, which can be naturally high or can be
toxicity in humans is the brain and that the developing brain increased by industrial emissions or the application of
is uniquely susceptible to MeHg neurotoxicity. Since then, contaminated fertilizing agents. It is not surprising, there-
evidence has emerged that much lower levels of prenatal fore, that cereals, tubers, and pulses were found to make the
MeHg exposure can affect human neurodevelopment, highest contribution (37–50%, depending on the age group)
resulting in altered motor function as well as memory and to Cd exposure in a Spanish population [41], whereas rice
learning deficits that may persist at least into adolescence. was a major determinant of Cd intake in a Japanese
These effects were particularly obvious in a large prospec- duplicate diet study [48]. Note, however, that by far the
tive birth cohort assembled in the Faroe Islands, where highest concentrations are found in fish and shellfish [41].
mothers are exposed to high levels of dietary MeHg due to In duplicate diet studies in an industrial and a nonindustrial
the occasional consumption of pilot whale meat [45]. Of area in Germany, children (mean age 3.8 years) were found
note, this population is also exposed to very high to have a Cd intake of 0.49 and 0.37µg/kg bw per day,
environmental levels of polychlorinated biphenyls (PCBs), respectively [49]. Adults from the industrial area had a
which themselves have been shown to affect cognitive mean intake of 0.37µg/kg bw per day. Maximum intake
development. There are indications that the effects of reached 120% of the current PTWI of 7µg/kg bw per week.
MeHg can be enhanced at the highest levels of PCB Other intake data are summarized in Table 5. In addition,
exposure [46]. Note, however, that PCB exposure was data from the NHANES III (1999–2000), which is
adjusted for in the Faroe Island cohort [45]. Some cross- representative of the US population 6 years and older,
sectional studies also found an association between prenatal indicate that even the 95th percentile of urinary Cd
MeHg exposure and neurobehavioral outcomes. In contrast, excretion is below 1.5µg/g creatinine [43]. This is well
another large longitudinal study from the Seychelles Islands below the level of 10µg/g creatinine that was thought to
did not yield any indications of such an association. Since represent a threshold for the occurrence of mild kidney
the two longitudinal studies have been used by various dysfunction manifested as reversible low-molecular mass
regulatory agencies in setting tolerable intakes, the inter- proteinuria. However, this threshold has recently been
pretation and weighting of the respective results have had a revised down to 1µg/g creatinine (see below) [50] and
major influence on the resulting values, which are none- that value is exceeded at the higher end of exposure in the
theless all in the range of 0.1µg/kg bw per day (USEPA) US population, even in adolescents (12–18 years of age)
and 0.3µg/kg bw per day (ATSDR), while the USFDA still [43].
lists an ADI of 0.4µg/kg bw per day based on data from the Cd is not well absorbed: the inhalation, oral, and dermal
Japan and Iraq poisoning incidences [47]. The FAO/WHO routes are associated with absorptions of 25%, 1–10%, and
<1%, respectively. Generally, females absorb a higher shellfish being in the inorganic form. As is well absorbed
proportion than do males. Cd is widely distributed from the small intestine, extensively methylated, and, to a
throughout the body, but the highest levels are found in lesser extent, conjugated to glutathione and quite rapidly
liver and kidney. It is not known to undergo any metabolic excreted, mostly in urine. Therefore, the low dietary intakes
conversion and is eliminated very slowly, with 0.007% and of mostly organic As reported from around the world (see
0.009% of total body burden being excreted daily via urine Table 5) indicate that food per se does not pose a substantial
and feces, respectively. Despite its low absorption, this risk in the USA or other industrialized countries. In
extremely limited elimination results in the life-long contrast, inorganic As is acutely toxic, with nausea,
accumulation of Cd in the kidney. vomiting, and abdominal pain constituting the initial
The IARC has classified Cd as a human carcinogen symptoms followed by psychosis, toxic cardiomyopathy,
(group I), but the evidence from human studies showing an seizures, and possibly death. In addition, high sublethal
association between Cd exposure and cancer of the lung, levels of inorganic As in drinking water result in chronic As
urinary bladder, kidney, and prostate is not deemed entirely toxicity characterized by keratosis and hyperpigmentation
convincing (even by the IARC itself). There is, however, of the skin, hepatomegaly, various neurological effects, in
strong evidence that Cd dose-dependently induces kidney particular peripheral neuropathy, both restrictive and ob-
tubular damage and a decrease in glomerular filtration rate structive lung disease, hematological abnormalities, and
that becomes irreversible at higher levels of exposure. The increased risk of cardiovascular and cerebrovascular disease
FAO/WHO set a PTWI of 7µg/kg bw (which would and diabetes. There also is a strong association between As
correspond to 60µg/day or 420µg/week for a person exposure and cancer of the skin, lung, and bladder, but so
weighing 60 kg). This is supposed to represent an intake far no studies from the USA have reported an increased risk
that can be maintained over a lifetime without resulting of these cancers in association with the drinking water
in Cd concentration in the renal cortex of more than levels of As. The WHO has set a PTWI of 15µg/kg bw per
200 mg/kg, a concentration at which tubular dysfunction week, corresponding to 2.14µg/kg bw per day.
occurs. In contrast, the USEPA has an oral Rfd for Cd
from food of 5µg/kg bw per day, i.e., 35µg/kg bw per
week. The European SCF initially endorsed the value set Organochlorine compounds
by the FAO/WHO. Recently, however, its successor
established a new TWI of 2.5µg/kg bw [50]. For its risk A variety of pesticides belong to the organochlorine group
assessment, it used urinary Cd levels as an indicator of of compounds, including 1,1,1-trichloro-2,2-bis(p-chloro-
body burden and urinary β2-microglobulin excretion as a phenyl)ethane (dichlorodiphenyl dichloroethene) (DDT),
marker of tubular effects. A one-compartment model was lindane, and other hexachlorocyclohexanes, cyclodienes
then employed to determine that an increase of urinary Cd such as dieldrin, chlordane, and heptachlor, and hexachlor-
of 4µg/g creatinine was associated with a 5% increase in obenzene. Their production and use in the USA were
the prevalence of elevated β2-microglobulin. To account banned as early as 1965 for hexachlorobenzene, in 1972 for
for interindividual variability in urinary Cd excretion, a Cd- DDT, and in the late 1980s for most other compounds.
specific adjustment factor of 3.9 was applied, indicating However, it was only in 2006 that the EPA called for
that urinary Cd concentrations should not exceed 1µg Cd per voluntary cancelation of products containing lindane, and
gram creatinine. Based on the analysis of dietary Cd intake the last use date for lindane was not until July 1, 2007.
and urinary excretion data from a large nonsmoking cohort, Many organochlorines, in particular the more heavily
it was then established that dietary intake of 0.36µg Cd/kg chlorinated ones, are lipophilic and very resistant to
bw per day (or 2.52µg/kg bw) over 50 years would lead to a degradation. Both factors contribute not only to their
urinary Cd concentration not exceeding the critical value of bioaccumulation in all parts of the environment but also
1µg/g creatinine determined from the preceding analysis, to their bioconcentration from one trophic level to the next.
hence the TWI of 2.5µg/kg bw. In this section, we focus on DDT and its breakdown
product dichlorophenyl dichloroethylene (DDE) as exam-
Arsenic (As) This element occurs naturally in inorganic ples of the toxicities associated with organochlorine
form (mostly arsenite As2O3 and arsenate As2O5, with pesticides.
AsIII being 60 times more toxic than AsV), and high levels
of inorganic As are found in certain water supplies, DDT and DDE The major route of human exposure to
particularly in India and Bangladesh, but also in certain DDT and its metabolite DDE is thought to be dietary
areas of the USA and of other parts of the world. The vast ingestion. DDT is well absorbed (70–80% depending on the
majority of As in food is in the organic relatively nontoxic matrix in which it is present). It is stored mostly in adipose
form, with ≤1.5% of As in fish and 10–20% of As in tissue, and humans retain DDT much longer than any other
species examined to date, including monkeys. The half- children of a birth cohort from Oswego, New York [54,55].
lives of DDT and DDE in humans have been estimated In contrast, cord blood concentrations of DDE in neonates
to be 7 and 10 years, respectively. Nonetheless, exposure of mothers who lived near a PCB-contaminated harbor in
and body burden have been steadily declining in Massachusetts were negatively associated with some items
populations from countries where DDT is not used for on the Neonatal Behavioral Assessment Scale relating to
malaria control. attention [56]. Equal or stronger associations were seen
The majority of the available data indicate that DDT and with PCB exposure, and there was no attempt to correct for
DDE are not genotoxic, although conflicting results have possible confounding (nor for the multiple comparisons).
been obtained. However, both are carcinogenic in animals, Both DDT and DDE were included in an investigation of
and the IARC has classified them as probably (group 2B) the CHAMACOS birth cohort from the Salinas Valley in
human carcinogens. Studies in human cohorts with rela- California. This cohort mainly included mothers who had
tively high exposures provide strong evidence for an lived in Mexico for most of their lives, and whereas DDT
association between DDE and testicular germ cell tumors was essentially banned in the USA in 1973, DDT use for
and DDT and liver cancer. The data on other types of malaria control continued in Mexico until the year 2000.
cancer are controversial and insufficient to determine The mothers’ serum concentrations of DDT and DDE
whether DDT and its metabolites are associated with (obtained during pregnancy) were not associated with
increased risk. An association between DDT and/or DDE behavioral assessment scores in their neonates [57] but
and type 2 diabetes has been observed in several studies but were associated with decreased scores in psychomotor and
may be confounded by concurrent exposures to other mental development assessments in their children examined
organochlorines. at 6, 12, and 24 months of age [58]. Specifically, DDT was
Studies in laboratory animals document that gestational associated with the psychomotor development index at 6
exposure particularly to DDE exposure during gestation has and 12 months, but not 24 months, whereas DDE showed
antiandrogenic effects. This is consistent with its ability to an association only at 6 months. The mental development
bind to the androgen receptor (AR) and inhibit androgen index was not related to DDT or DDE exposure at 6 months
binding. In contrast, o,p′-DDT, a minor component of but was inversely correlated with both compounds at 12
technical-grade DDT, and some DDT metabolites exhibit and 24 months. Note that DDE and DDT were highly
estrogenic activity. The effects of DDE on the male correlated in this cohort, making it difficult to separate their
reproductive organs include reduced weight of testes, effects. However, the results were independent of possible
seminal vesicles, glans penis and cauda epididymis, neurodevelopmental effects of PCBs, Pb, organophosphate
decreased sperm count and motility, reduced anogenital (OP) pesticides, or hexachlorobenzene. In another group of
distance, and nipple retention. In rabbits, a low incidence of Mexican women from an area with endemic malaria,
cryptorchidism was also observed. In adult animals, DDT maternal DDE levels during the first trimester of pregnancy,
exposure can result in reduced testosterone production. but not those obtained during the second or third trimester,
Several studies in adult men suggest an association between showed a significant inverse association with the psycho-
DDE and/or DDT exposure and semen quality, but the motor development index (but not the mental development
findings are not consistent. In women, there are indications index) on the Bayley Scales for Infant Development in their
that DDT and DDE exposure is associated with alterations infants at 3, 6, and 12 months of age [59]. Too many of the
in estrogen and progesterone levels at different phases of samples contained undetectable levels of DDT to allow
the menstrual cycle, and this may represent an increased analysis of an association between this compound and
risk for fetal loss. There are several reports that the levels of neurodevelopment.
DDT/DDE exposure seen during the peak usage of this In two Spanish birth cohorts, gestational DDT exposure,
pesticide in the 1950s and 1960s were associated with an as assessed by DDT concentrations in cord blood, was
increased risk of preterm birth, being small for gestational significantly associated with decreased general cognitive,
age, and having reduced birth weight. More recent data memory, and verbal scores in the McCarthy Scales of
suggest that these effects are no longer seen at current Children’s Abilities [60]. This association remained after
reduced exposure levels. adjustment for DDE concentrations, whereas the only
In recent years, more information has become available significant association seen with DDE (decreased memory
on the possible association of DDT or its breakdown scores) disappeared after adjustment for DDT exposure.
product DDE and neurodevelopmental outcomes. In a birth To summarize, data on associations between prenatal
cohort from North Carolina, transplacental exposure to exposure to the parent compound DDT and neurodevelop-
DDE was associated with hyporeflexia in neonates [51] but mental outcomes are limited but show a consistent negative
did not affect later behavioral or cognitive development effect on mental and behavioral development that is
[52,53]. A similar lack of association was described in independent of other exposures implicated in disturbed
early cognitive functioning. While more data exist on the result by the concentration of the individual dioxins and
possible effects of gestational exposure to the breakdown dioxin-like compounds in the diet. Dietary exposure and
product DDE and neurodevelopment, the results are body burden have been declining over the last decades.
conflicting. Some of these discrepancies may be due to Relatively current mean intake estimates range from
different exposure levels but also to differences in the 0.38 pg TEQ per kilogram bw from PCDD/Fs for the US
analytical procedures (cord blood versus maternal serum) population and from 0.4 to 1.5 pg TEQ per kilogram bw
and the control for covariates in the statistical analysis. per day in various European countries, with PCBs adding
another 0.8–1.5 pg TEQ per kilogram bw per day. The total
Dioxins and dioxin-like compounds In addition to DDT and exposure in Europe therefore amounts to 1.2 to 3 pg TEQ
other pesticides, the organochlorines include polychlori- per kilogram bw per day. That indicates that a substantial
nated dibenzofurans (PCDFs) and dibenzodioxins portion of the population is exposed to levels higher than
(PCDDs), PCBs and polybrominated biphenyls (PBBs), the TWI of 14 pg TEQ per kilogram bw set by the JECFA
and polychlorinated naphthalenes. Whereas PCBs and and other regulatory agencies. Children generally are
PBBs were purposely produced for use in transformers exposed to considerably higher amounts of dioxins and
and capacitors, hydraulic fluid, plasticizers, and fire dioxin-like compounds. In the USA, children at the age of
retardants, PCDD/Fs are byproducts of thermal and 2 years had a mean intake of PCDD/Fs of 1.2 pg TEQ per
industrial processes, now stemming mostly from incinera- kilogram bw per day, and European studies also indicate
tion of municipal and hazardous waste. According to that the dietary exposure of children is up to threefold
conservative estimates, ~1.3 million tons of PCBs were higher than that of adults. Note that it is deemed advisable
produced, almost exclusively in the Northern hemisphere. to include polychlorinated naphthalenes in the TEQ
Some of the major PCB congeners were recently estimated scheme, but sufficient data for comparing its potency to
to have environmental residence times of 70–100 years. that of TCDD are not yet available [62]. In one of the rare
This suggests that, even though the production of PCBs studies examining exposure to polychlorinated naphtha-
was halted ~30 years ago, exposure will continue for lenes, mean dietary intake of this compound was 0.1 ng/kg
decades, if not centuries. bw per day in a Spanish population, which represented an
Potentially, there could be 75 different PCDD and 135 80% decrease compared to results obtained in 2000 [63].
PCDF congeners (isomers with similar halogen substitution Of particular note, dietary exposure to PCDD/Fs and
patterns), but the actual number present in biotic samples is PCBs starts in utero as evidenced by their detection in
much lower, with mainly 2,3,7,8-substituted congeners amniotic fluid and cord blood. Even though breast milk
being detected. The most toxic congener is 2,3,7,8- levels have been declining over the last decades in most
tetrachlorodibenzo-p-dioxin (TCDD), which is often re- industrialized countries, infant exposures from breast
ferred to simply as “dioxin.” The term “dioxins” refers to feeding still frequently exceed the most commonly used
PCDDs in general. Although 209 PCB congeners are TDI values by two to three orders of magnitude. Such
possible, only between 50 and 150 congeners are detectable values are based on lifetime intakes and not intended to be
in biotic samples [61]. Whereas PCDDs and PCDFs have applied to the relatively short nursing period. On the other
rigid planar structures, PCB molecules can be more hand, the exposure during nursing has been found to be a
flexible, depending on the number and positions of the determining factor of total body burden well into adoles-
chlorine substituents. The least-flexible, planar PCBs cence. In addition, infants are likely to be considerably
exhibit the greatest resemblance with the dioxins and are more susceptible to the various toxic effects of environ-
often referred to as “dioxin-like” compounds. mental pollutants, including organochlorine compounds.
Like DDT and DDE, dioxin and dioxin-like compounds Although only limited data are available, it is generally
accumulate in the environment and are bioconcentrated in accepted that humans readily and almost completely absorb
higher trophic levels of the food chain. There are lower-chlorinated PCB and PCDD/F congeners, while
indications that ~90% of current human exposure to PCBs, absorption of higher chlorinated congeners is lower but
dioxins, and dibenzofurans occurs via dietary intake of still substantial. Based on animal studies, dermal absorption
contaminated foods. The highest levels of contamination is estimated to range from 8% to 20%, depending on the
are usually found in foods containing animal fats, such as degree of chlorination and on methodological factors.
meat, dairy products, and fish, particularly fish from highly PCBs are initially distributed to highly perfused tissues
contaminated waters like the Great Lakes or the Baltic Sea. such as liver and muscle but are then redistributed to and
The results of exposure assessment are expressed in toxic stored in adipose tissue and skin. In partial contrast, >95%
equivalent (TEQ), which are derived by determining the of the body burden of PCDD/Fs is found in the liver and
potency of PCDDs, PCDFs, and certain PCBs relative to adipose tissues. Many PCBs and dioxins have long half-
the most toxic compound, TCDD, then multiplying the lives in humans, with estimates of ≥30 years for the more
persistent PCBs, 7 or 8 years for TCDD, >15 years for changes in the sex ratio were found in the children of three
other PCDDs, and up to 20 years for some PCDFs. The other cohorts exposed to high levels of PCBs, PCDFs, and
major metabolites of PCBs are methyl sulfones and thermal degradation products of these compounds. Unlike
polychlorobiphenyls (OH-PCBs). Some OH-PCBs are in laboratory animals, exposure to background levels of
selectively retained, mainly by binding to plasma proteins, PCB, PCDDs, and PCDFs is not consistently associated
such as albumin and the thyroid hormone transport protein, with negative effects on birth outcomes or thyroid function
transthyretin (TTR). In vitro, certain OH-PCBs have [64].
fourfold higher affinity for TTR than its natural ligand In children from a Dutch birth cohort examined at the
thyroxine, and their ability to interfere with thyroid age of 42 months, higher prenatal, but not postnatal,
hormone homeostasis may contribute to the neurodevelop- exposure to PCBs was associated with subtle changes in
mental effects of PCB exposure. lymphocyte subset distribution and with decreased levels of
serum antibodies to mumps and rubella [65]. Similarly, in
Health risks of dioxins and dioxin-like compounds routinely vaccinated children from two Faroe Islands birth
cohorts, there was an inverse association between prenatal
In animals, dioxins and dioxin-like compounds exhibit a PCB exposure (assessed as maternal serum concentrations)
broad array of toxicities, ranging from disturbances of and serum antibody levels for diphtheria toxoid at
multiple hormone systems and toxicities of the liver, the 18 months and for tetanus toxoid at 7 years of age [66].
developing immune, nervous, and reproductive systems to Postnatal exposure (the child’s own serum PCB concentra-
carcinogenesis and outright lethality. There are marked tion at the time of examination) showed similar associa-
interspecies differences in the susceptibility to dioxin tions, and early postnatal exposure in particular was an
lethality and certain other outcomes, whereas some effects important predictor of diphtheria antibody levels at
are seen at similar body burden in essentially all species 18 months of age. In the Dutch cohort, increased postnatal
examined to date. The immune system, particularly during PCB exposure (current serum PCB concentration) was
fetal development, represents one of the most sensitive associated with a higher incidence of otitis media, whereas
targets of TCDD, other dioxins, and dioxin-like com- gestational exposure was associated with less shortness of
pounds. Gestational or perinatal exposure results in thymic breath with wheeze. An association between perinatal PCB
atrophy at relatively high doses, but even low doses lead to exposure and otitis media was also observed in some Inuit
altered structural and functional development of the cohorts, along with an increased frequency of upper-
immune system and permanent suppression in delayed- respiratory infections, gastrointestinal infections, and infec-
type hypersensitivity. In adult laboratory animals, including tious episodes overall. Although these findings suggest
nonhuman primates but also in marine mammals, chronic subtle effects on the developing immune system with
low-dose exposure to dioxins suppresses both humoral and possible clinical relevance, the results need to be interpreted
cell-mediated immune responses and is associated with with great caution due to a variety of methodological
impaired host resistance to various infectious diseases. shortcomings [64]. Few studies have examined the immu-
Another highly sensitive target is the developing brain. nological sequelae of dioxin and PCB exposure in adults.
Gestational exposure of rodents and monkeys to PCBs Although there are occasional reports of disturbed lympho-
consistently results in negative effects on learning and cyte subset distribution and decreased serum concentrations
locomotor activity and function [61]. of immunoglobulin and complement, the results are highly
The IARC has classified TCDD as a human carcinogen inconsistent and do not provide convincing evidence of
(group 1) but considered other PCDDs and PCDFs as not immunotoxicity.
classifiable. In occupationally and otherwise highly ex- One of the greatest concerns over the continuing human
posed cohorts, TCDD and possibly other PCDD/Fs are exposure to PCBs and PCDD/Fs are their possible neuro-
associated with increased mortality from ischemic heart developmental toxicities. Children of mothers exposed to
disease, but this is not an entirely consistent finding. There high levels of PCBs, PCDFs, and thermal degradation
are also indications that even background levels of TCDD products of these compounds due to the ingestion of highly
exposure may increase the risk of type 2 diabetes, but such contaminated cooking oil displayed developmental delay
an association was not detected in other studies. Higher and impaired performance on behavioral and cognitive
levels of paternal exposure to TCDD stemming from an function tests. Numerous cohort studies have examined the
industrial accident in Seveso, Italy, were found to be effects of prenatal exposure to environmental background
associated with a decreased male-to-female sex ratio in levels to PCBs on neurodevelopmental outcomes. Exposure
their children. Similar observations were reported from was assessed as maternal or infant body burden, as reflected
workers from a Russian pesticide-producing plant exposed by concentrations in maternal serum or in cord blood.
to high levels of dioxin. On the other hand, no significant Overall, the results of these studies indicate that prenatal
PCB exposure is associated with subtle but significant The JECFA has set ADIs of 0–0.05µg/kg bw per day for
delays in the early neurodevelopment of infants and estradiol 17β, 0–30µg/kg bw per day for progesterone, and
children [67]. In some cohorts, these effects were persistent 0–2µg/kg bw per for testosterone. Assuming a theoretical
until at least 7 years of age, whereas in others they had daily meat intake of 500 g (300 g muscle, 100 g liver, 50 g
disappeared as early as the age of 18 months. Note that fat, 50 g kidney—which seems a very high estimate), it has
PCB exposure was not associated with neurodevelopmental been calculated that the contribution to the daily maximum
outcomes in some of the most recent cohorts [68,69], and it intake of estradiol of 3µg (FAO/WHO ADI of 0.05µg/kg
has been suggested that this is related to the slow decline in bw in a 60-kg adult) is 0.2% from untreated animals, 1.3%
exposure and body burden seen over recent decades. from animals treated according to the existing guidelines,
Despite the much greater transfer of organochlorines from and up to 6.7% from animals with off-label use (two
the mother to the infant via breast milk, most studies do not implants) [73]. Note, however, that currently used methods
reveal any significant associations between postnatal are not designed to detect estradiol fatty acid esters, and it is
exposure via breastfeeding and neurodevelopmental out- somewhat unclear to what extent glucuronide and sulfate
comes, although there are some notable exceptions. conjugates are hydrolyzed before analysis and subsequently
Nonetheless, WHO did not find sufficient evidence to measured [70]. Estradiol fatty acid esters have been found
change its endorsement of breastfeeding. to exert stronger estrogenic effects compared to estradiol-
17β after oral administration, possibly because of slower
more sustained absorption and higher bioavailability [74].
Substances arising from production processes There are indications that the metabolism of trenbolone,
melengestrol acetate, and zeranol yields a metabolite profile
Hormones that is much more complex than previously thought. The
biological activity of these metabolites has not been
Six steroid hormones are approved by the USFDA, namely thoroughly investigated. Given these data gaps, exposure
estradiol, progesterone, testosterone, zeranol, trenbolone and risk assessment of hormone and synthetic growth
(acetate), and melengestrol acetate. The first three are promoter residues in the meat of treated animals is currently
natural female and male sex hormones, respectively. impossible. This would be highly desirable, however, given
Zeranol is a synthetic form of α-zearalenol, an estrogenic the increasing evidence that estrogens are carcinogens.
metabolite derived from the mycotoxin ZEA. Trenbolone The issue of whether hormonal residues in meat,
and melengestrol acetate are synthetic anabolic steroids. particularly beef, are a human health concern has led to a
In the Red Book containing data on 2007 surveillance major trade dispute between the USA and Canada on one
activities of the FSIS/USDA, it was reported that testing for hand and the EU on the other hand because the EU refuses
the three synthetic growth promoters did not reveal any to allow the import and sale of meat from hormone-treated
residue violations in 261 veal samples tested for zeranol, animals. In addition to the limited knowledge about
258 veal samples tested for trenbolone, and 309 heifers metabolites and their relative potencies, the EU Scientific
tested for melengestrol acetate. No nonviolative positives Committee on Veterinary Measures raised the issue of
were seen for zeranol or trenbolone, but two samples tested incorrect or off-label use. They deemed such use a common
positive at nonviolative levels for melengestrol acetate. The occurrence based on statements made by representatives of
published literature on the tissue levels of hormones in the US beef industry and on numerous scientific publica-
animals treated with one of the natural or synthetic growth tions advocating multiple dosing. They further feared that
promoters indicates that the responses of individual animals incorrect placement of the hormonal implants could result
are highly variable but that there generally is a dose- in their being overlooked at slaughter and could result in
dependent increase in parent compound and metabolites. contamination levels of whole batches of meat that would
The magnitude of this increase strongly depends on the far exceed the MRL and, if they reached the customer,
compound or metabolite and the tissue examined, for might be high enough to cause acute hormonal effects.
example ranging from 3.7 in muscle to 6.4 in fat for Even if it could be assumed that hormone residues at the
estradiol-17β and from 1.2 in muscle to 1.9 in fat for estrone current tolerances and their metabolites are by themselves
in animals with estradiol-containing implants [70]. Similar insufficient to affect human health, the question remains to
results have been reported for animals treated with other what extent they interact with the numerous other hormone-
growth promoters [71,72]. Note that zeranol, i.e., α- like substances we have been and continue to be exposed to
zearalenol, is a minor metabolite in the tissue of untreated from food (and from the endogenous burden we have
pigs if their feed is contaminated with the mycotoxin ZEA, accumulated over a lifetime) and whether eliminating an
whereas it was found to be undetectable in muscle or liver entirely avoidable source of hormones would not be a good
tissue of cattle that had received ZEA-contaminated feed. place to start reducing this exposure.
Antibiotics and other veterinary drug residues development of new antimicrobial agents. Veterinary anti-
biotics are used not only for veterinary indications, i.e., to
The USFDA and similar regulatory agencies in many other treat infections, but are routinely added to animal feed to
countries require premarket safety and efficacy testing of increase feed efficiency and enhance growth in the USA
veterinary drugs analogous to pharmaceuticals for human and other countries. The EU banned all uses of antibiotics
usage. In addition, in order to ensure the safety of meat as growth promoters in 2006. Although in most countries,
products from drug-treated animals, the producers of antimicrobials used as performance enhancers must not
veterinary drugs have to provide evidence that residues in include products that are used for therapeutic purposes in
such meat products do not cause harm to humans. These humans and/or animals, they frequently are analogs of
data are then used not only to establish ADIs where therapeutic antibiotics and show cross-resistance. There are
applicable and to set tolerances or MRLs but also to numerous lines of evidence strongly suggesting that the
determine withdrawal times. The withdrawal time is the high level of antibiotic use in animal husbandry is a major
period that is required for the elimination of the antibiotic contributor to the emergence, selection, and dissemination
from the animal’s tissue, i.e., the time that has to elapse of resistant bacteria [75]. As in humans, it has been shown
after the last administration of a particular veterinary drug that the introduction of a specific antibiotic into veterinary
before the animal can be slaughtered. In the USA, practice, particularly as an antimicrobial performance
tolerances for veterinary drug residues are listed in the enhancer, results in an increase of resistance to this
Code of Federal Regulations 21CFR 556. The USA, the antibiotic not only in pathogenic bacteria but also in the
EU, and Japan agreed to harmonize their legislation on bacteria of the intestinal microflora of the treated animals. It
the requirements for registration of veterinary medicinal has further been demonstrated that such resistant bacteria
products. This has resulted in a series of new guidelines can be transferred to humans either by direct contact with
from the Center of Veterinary Medicine of the USFDA the animal, with its fecal matter or with meat products.
(www.fda.gov/cvm/guidance/published.htm) providing There are also indications that resistance genes can be
details on the genotoxicity, carcinogenicity, and develop- transferred between pathogenic bacteria and residents of the
mental toxicity testing required in the safety evaluation of intestinal microflora. Thus, the intestinal microflora con-
veterinary drugs. Note that the intestinal microflora has stitutes an important reservoir of resistance genes and every
important functions in preventing the colonization of the effort should be made to keep this reservoir as small as
gastrointestinal tract with pathogenic bacteria and in possible. It has been estimated that the EU ban on
stimulating the gut-associated immune system. The effects antimicrobials as performance enhancers would reduce
of antibiotic residues in meat on the human intestinal antibiotic use in animals by 30–50%. And since 50% of
microflora seem to have been rarely considered in earlier all antibiotics used in the EU were given to animals before
toxicological testing of veterinary drugs but are now their ban as growth promoters, this would result in a
included in the new harmonized guidelines. considerable reduction of antimicrobial use overall [75].
The FSIS/USDA is responsible for inspecting domestic The USFDA also is concerned that human exposure to
and imported meat for veterinary drug residues. As antibiotic-resistant bacteria via the ingestion of animal-
illustrated in the Red Book published by the FSIS, the derived products could reduce or destroy the effectiveness
detection of veterinary drug residues at levels exceeding of antimicrobial drugs used to treat human infections.
their respective tolerances is a rather rare occurrence. For Another one of the new USFDA guidance therefore deals
example, only four of 3,372 domestic samples contained with the types of premarket tests of new veterinary
antibiotic residues at levels exceeding the tolerance, while antimicrobials that are required in order to minimize this
193 (5.7%) were positive but did not violate existing risk.
guidelines. This suggests that exposure to antibiotic and
other veterinary drug residues is unlikely to cause direct
health effects in human consumers. Pesticide residues
However, another more indirect effect on human health
needs to be considered in the case of antimicrobial drugs. Organophosphate pesticides
Antibiotic resistance has become a major cause for concern
because there are now some microorganisms that are The term pesticide is used to refer to herbicides, insecti-
resistant to (almost) every antibiotic currently used in cides, fungicides, fumigants, rodenticides, and other chem-
human medicine, making it increasingly difficult—and icals designed to destroy or repel pests. According to data
expensive—to treat certain diseases. It is feared that this published by the USEPA, their use (as active ingredient at
development may reach a point where the emergence of user level) exceeded five billion pounds worldwide and 1.2
antibiotic resistance in pathogenic bacteria outpaces the billion pounds in the USA in each of the years 2000 and
2001. When chlorine/hypochlorites (2.5 billion pounds), estimates of total dietary OP exposure based on probabilistic
wood preservatives, and specialty biocides were included, approaches indicated that the median exposure to OP (in
total usage in the USA alone was almost five billion acephate equivalents) and carbamate pesticides (in oxamyl
pounds. At least 75% of this amount is used in agriculture, equivalents) was zero for adults and children. At the 99th
while the remainder is equally divided between home and percentile of exposure, adults consumed 3.9µg OP per
garden use and commercial/industrial/government. Appli- kilogram bw per day and 0.11µg carbamates per kilogram
cation of OP pesticides declined by nearly 45% between bw (analogous to 273 and 7.7µg/day in an adult weighing
1980 and 2001 but still amounted to 73 million pounds in 70 kg) [77]. The corresponding figures in children aged
2001. This is the first group of pesticides we focus on in 1–6 years were 7.9µg OP per kilogram bw per day and
this section because it has been the focus of much 0.28µg carbamates per kilogram bw per day.
regulatory attention in recent years, as will be discussed Pharmacokinetics studies in human volunteers indicate
in more detail in the last section of this review. that OPs are readily absorbed after oral administration and
Exposure to pesticides can occur via inhalation, dermal quickly metabolized to more polar metabolites, which are
absorption, and ingestion. What limited data are available then eliminated in urine with half-lives ranging from 2 h for
on the subject suggest that inhalation makes by far the orally administered diazinon up to 27 h for chlorpyrifos.
greatest contribution to aggregate exposure of certain OP Therefore, biomonitoring potentially provides a method of
compounds (~85%) in adults, with much of the remainder estimating total exposure to OP pesticides, either by
stemming from pesticide residues in solid food. In children, measuring urinary metabolites or by quantifying concen-
however, solid food may constitute the major route of trations of the pesticides themselves and/or some of their
exposure. In contrast, in their assessment of aggregate metabolites in plasma. Problems associated with the
(from all exposure pathways and routes) and cumulative measurement of urinary OP metabolites include that (a)
(from all OP pesticides) exposure to OP pesticides, the only some of the pesticides in common use have specific
USEPA concluded that food made the major contribution to metabolites whereas dialkyl phosphate (DAP) metabolites
overall risk in the population in general. However, their are nonspecific since they can be derived from a wide
data also indicate that whether food, other oral exposure variety of OP compounds; (b) data on the fractional
(e.g., hand-to-mouth behavior of small children), drinking absorption and excretion are largely unavailable; and (c)
water, or inhalation from residential application dominat- urinary metabolites can arise either from a parent com-
ed total exposure depended on the age group examined, pound or from direct exposure to its metabolites. Further-
geographic region, and time of year and whether the more, unless 24-h urine samples are obtained, urinary
95th, 99th, or 99.9th percentile was considered. Within metabolite concentrations need to be corrected for dilution,
the category of residential exposure, inhalation repre- but the appropriate method is still a matter of debate.
sented the major route of exposure. Note that the USEPA OP pesticides irreversibly inhibit the enzyme acetyl
considered only residential exposure arising from the cholinesterase (AChE). Inhibition of AChE at neuronal
application of pesticides in the home or garden or from junctions results in the accumulation of acetyl choline
pet collars but did not take into account that residents of (ACh) and continued neurotransmission. Since the auto-
homes situated near agricultural areas are exposed to nomic, somatic, and central nervous system all use ACh,
significantly higher environmental and residential levels the acute symptoms of OP-mediated AChE inhibition are
of pesticides, including OP pesticides [76]. manifold and include dizziness, headache, confusion,
Based on food consumption data from two large cohort convulsions, blurred vision, respiratory distress, bradycar-
studies combined with the data from the USFDA Total Diet dia and hypotension, fatigue, weakness, ataxia, muscle
study, it was estimated that mean daily dietary intakes of cramps, and increased lacrimation and salivation.
chlorpyrifos, diazinon, and malathion were 0.8, 0.5, and
5.5µg/day for women and 0.9, 0.5, and 6.1µg/day for Neurodevelopmental toxicities In experimental animals,
men. Analysis of duplicate diet samples indicated an gestational or early postnatal exposure to OP pesticides at
adult dietary chlorpyrifos and malathion intake of 0.5 and levels that inhibit AChE to a minor extent (~20%) and are
1.3µg/day, respectively, and a dietary chlorpyrifos intake not associated with overt systemic toxicity results in
in children of 0.263µg/day. The mean dietary intake of total impaired neurodevelopment. In addition to brain AChE
OP and carbamate pesticides expressed in chlorpyrifos and choline acetyltransferase inhibition, a variety of
equivalents was estimated to range between 5.7 and mechanisms may contribute to the neurodevelopmental
13.6µg/day in the average adult Dane, depending on effects of OP pesticides. These include alteration of
whether samples below the detection limit were set to zero muscarinic receptor function and permanent reduction in
or one half the limit of quantitation. The corresponding the density of muscarinic cholinergic receptors, altered
values in the average child were 3.9 to 9.3µg/day. Recent synaptic development and function that can persist into
adulthood, decreased expression and activity of multiple cognitive function despite the potentially higher pesticide
components of the adenylyl cyclase cascade, impaired residue intake. Most importantly, however, it needs to be
DNA and RNA synthesis, and reduced cellularity and brain taken into account that spot urine samples do not provide a
weight in offspring. good estimate of chronic pesticide exposure, as shown by
The detection of pesticides or their metabolites in human the high day-to-day variability in most biomonitoring
amniotic fluid and meconium and strong correlations studies.
between OP pesticide/metabolite concentrations in maternal Of particular note, exposure to a variety of other
and umbilical cord plasma indicate that human exposure to compounds known or suspected to affect neurodevelop-
these compounds can begin in utero. In recent years, the ment was measured and controlled for in the CHAMACOS
neurodevelopmental effects of OP pesticide exposure have study, including PCBs, β-hexachlorocyclohexane, hexa-
been examined in several birth cohorts that recruited chlorobenzene, DDT, and its breakdown product DDE [81].
mothers with high levels of exposure due either to frequent There was no statistically significant interaction between
pesticide applications in the home (as in two cohorts from DDT and prenatal OP exposure (maternal urinary DAP
New York City) or to their extensive use in surrounding metabolites), indicating that the effects of the two expo-
agricultural areas (as in the CHAMACOS project in the sures are independent. This is of particular interest since a
Salinas Valley, CA, USA). previous study in the same cohort had revealed an
In neonates of the CHAMACOS cohort, maternal association between prenatal DDT exposure (maternal
urinary DAP metabolites were significantly associated with serum DDT levels) and mental development scores at 12
an increased number of abnormal reflexes (failure to and 24 months of age [58].
respond or hypoactive response) and with the proportion
of neonates with more than three abnormal reflexes [78]. Pregnancy outcomes There are several reports that OP
Interestingly, the association differed depending on the age pesticide exposure during pregnancy (as assessed by
at the time of the behavioral assessment. The association metabolite concentrations in one or two spot urine samples)
was negative in neonates examined after the age of 3 days is associated with decreased birth weight and length
but unexpectedly positive in infants assessed within the first [82,83]. In contrast, in the CHAMACOS cohort, maternal
3 days of life. Although this suggests some caution in the urinary DAP metabolites were associated with a significant
interpretation of these results, they were essentially repli- increase in head circumference and a marginally significant
cated in one of the New York cohorts, i.e., maternal urinary increase in birth length [84]. The dimethyl phosphate subset
concentrations of DAP metabolites as well as malathion of DAP metabolites, but not total DAP, and cord blood
dicarboxylic acid (a specific metabolite of malathion) were cholinesterase activity were significantly associated with
significantly associated with the number of abnormal decreased gestational duration.
reflexes in neonates [79].
At 3 years of age, children from New York City with
high levels of gestational OP pesticide (specifically chlor- Contaminants formed during food production
pyrifos) exposure were significantly more likely to exhibit and cooking processes
mental and psychomotor developmental delays, attention
problems and attention-deficit/hyperactivity disorder, and A variety of chemicals are formed in the food production
pervasive developmental disorder problems [80]. Note that process or during cooking. These include polycyclic
the children’s own exposure during the first 3 years of life aromatic hydrocarbons, which occur mainly in smoked
was not taken into account in this study. In the CHAMA- and grilled meat, i.e., in situations where combustion
COS cohort, gestational OP pesticide exposure was products come into direct contact with food. The highly
associated with lower scores in assessments of mental carcinogenic benzo(a)pyrene is a major representative of
development, the association reaching significance by the this group. In addition, heterocyclic aromatic amines are
age of 24 months, but not at 6 or 12 months of age [81]. No formed in heated meat products and have been implicated
association with attention problems was detected at any in causing mammary and colorectal cancer. Furthermore,
age. Interestingly, postnatal OP exposure (as assessed by nitroso compounds, such as N-nitrosamine, N-nitrosamide,
metabolites in spot urine samples obtained at 6, 12, and and related substances, result from the reaction of the
24 months of age) showed a positive association with preservatives nitrate or nitrite with secondary amines.
mental development scores. The authors hypothesized that Carnitine, choline, citrulline, creatine, creatinine, pyrroli-
children with higher cognitive functioning may have more dine, or trimethylamine all constitute relevant precursors for
interactions with their environment that would expose them this reaction in foods. Many of the nitroso compounds are
to higher pesticide levels or that better diets with higher potent genotoxic carcinogens in laboratory animals. In the
levels of fruits and vegetables may result in improved following, we focus on some other chemicals that arise
from cooking processes and that have been the particular lots of the same food because the formation of acrylamide
focus of risk assessment efforts in recent years. depends on cooking temperature, cooking time, and the
composition and other characteristics of the specific food.
Acrylamide (CH2 =CH–CONH2) is produced industrially to In addition, the analytical method has been found to have a
synthesize polyacrylamide, which is used as a soil significant influence on the test results.
conditioner, for water treatment, in grouting compounds, What foods are the main contributors to dietary
in gels used for electrophoresis, and numerous other acrylamide intake in a particular population depends on
applications in the paper, textile, and cosmetic industries eating habits and the way foods are processed and prepared,
[85]. In addition, it is present in cigarette smoke. In 2002, although a considerable portion derives from French fries
the Swedish National Food Administration and researchers and other potato products in most Western nations (e.g.,
from Stockholm University announced the discovery that 35% in the USA, 50% in the Netherlands, 30% in Norway,
acrylamide also can be formed when certain types of and 26% in Sweden) [87]. In contrast, acrylamide from
food are fried, deep-fried, or baked at high temperatures coffee constitutes only 7% of total dietary intake in the
(>140°C) [86]. Since then, it has become widely recog- USA but is the major contributor (39%) in Sweden.
nized that particularly high levels are present in French fries Acrylamide is well absorbed by ingestion, inhalation,
and potato chips. Considerable concentrations have also and after dermal application. Studies in mice and rats
been found in bread, cookies, crackers, cereals, and also in indicate greater bioavailability when administered as a
coffee, but lower levels of acrylamide can be detected in bolus by gavage than when given as part of the diet, which
numerous other foods. It is generally accepted that the is usually consumed over several hours. There are two
acrylamide in food is mostly formed from the reaction of major metabolic pathways: (a) conjugation to glutathione
asparagine with reducing sugars. More recently, acrylamide and (b) oxidization to its epoxide glycidamide, which in
has also been detected in prune juice and black olives, rodents is mediated by CYP2E1. Like acrylamide, glycida-
indicating that other pathways can lead to the formation of mide can be conjugated with glutathione. Both glutathione
this substance. Since the discovery of acrylamide formation conjugates are excreted in urine as mercapturic acids.
in foods, numerous western countries have collected data Urinary metabolites can be used to assess acrylamide
on the dietary intake of this chemical [87]. These exposure exposure over the last 24 h since acrylamide metabolites
assessments were based on national food surveys that were have half-lives of elimination between 14 and 26 h [89].
not specifically intended to assess dietary acrylamide Both acrylamide and glycidamide form hemoglobin adducts,
intake. The intake levels determined in these surveys were and these can be used to estimate total exposure over the last
then linked with data on acrylamide concentrations in a 2–3 months (the average lifetime of an erythrocyte). There
limited number of food groups known to contain high is fairly good agreement between studies in various
levels of this compound. Therefore, the resulting data need nonsmoking western populations that median excretion of
to be interpreted with some caution. The USFDA estimated urinary metabolites is approximately 30–40µg/l for acryl-
mean dietary acrylamide intake of adults in the USA to be amide mercapturic acid and 3–9µg/l for glycidamide
0.4µg/kg bw per day. This is in close agreement with mercapturic acid. Median hemoglobin adduct levels in such
estimates from other western nations, for example 0.2–0.4 populations range between 26 and 37 pmol/g hemoglobin
in EU countries overall, 0.5 in France, 0.45 in Sweden, or for acrylamide and between 18 and 34 pmol/g hemoglobin
0.48 in the Netherlands. The 95th (or 90th) percentile for glycidamide. Estimates of dietary intake calculated from
ranged from 0.6 to 1.1. Note, however, that the mean (and urinary metabolite excretion or hemoglobin adduct levels
90/95th percentile) daily intake of children is generally suggest median acrylamide exposures in a similar range as
estimated to be twofold to threefold higher, being 1.07 obtained in dietary survey studies, namely median levels of
(2.31) µg/kg bw per day in small children (aged 2–5 years) ~0.45µg/kg bw per day from urinary metabolites and of
in the USA. Other subpopulation may also have high 0.51µg/kg bw per day from hemoglobin adducts [90].
intakes, as indicated by the observation that the 15–18-year- Glycidamide is genotoxic, whereas acrylamide itself is
old age group in Germany had a mean (95th percentile) not, and glycidamide is thought to be responsible for the
dietary exposure of 1.1 (3.4) µg/kg bw per day. Using a carcinogenic effects of acrylamide exposure in laboratory
semiprobabilistic approach, the mean dietary exposure of animals. The IARC has classified acrylamide as a probable
the UK adult population was calculated to be 0.56µg/kg bw human carcinogen (group 2B). Recently, and particularly in
per day [88]. A probabilistic model yielded an estimate of the last few months, numerous studies have been published
0.59µg/kg bw per day in the Irish adult population [88]. on the possible association between acrylamide exposure as
These numbers provide only rough estimates since there is measured by dietary intake assessment and the risk of
considerable variability in acrylamide content between various cancers. For the most part, acrylamide exposure
products of the same category of food and even between was not found to be associated with cancer of the
gastrointestinal tract (colorectal, gastric, pancreatic, or that acrylamide is a neurotoxin, and this is also observed in
esophageal), brain, prostate, endometrium, or breast. One animals. However, the results of physiologically based
exception comes from a case–cohort that was part of a large pharmacokinetic/pharmacodynamic modeling based on
prospective Dutch cohort study, in which dietary intake of tissue concentration of acrylamide or its metabolite glyci-
acrylamide was associated with postmenopausal endome- damide indicated that the exposure levels arising from
trial and ovarian cancer, but not breast cancer, particularly dietary consumption of acrylamide were not associated with
in nonsmokers [91]. In the same cohort, the highest significant human risk [94].
compared to the lowest quintile of dietary acrylamide
intake conferred a slightly but significantly increased risk Acrolein Acrolein is an intermediate in the industrial
of renal cell but not bladder or prostate cancer [92]. Note production of acrylic acid but is also released in combustion
that many of these studies may have lacked the statistical processes (including tobacco smoking) and may be an
power to detect the small increase in cancer risk that may intermediate in the formation of acrylamide in foods. It is
be expected in association with acrylamide exposure. In formed from carbohydrates, vegetables oils, animal fats, and
addition, the wide distribution of acrylamide in foods and amino acids during the heating of foods. In particular, the
the high variability of its concentrations even within foods heating of cooking oil to temperatures >180°C generates
of the same category make exposure misclassification likely considerable amounts of acrolein, and it has been estimated
when dietary intake is used as a measure of exposure. In the that the emissions from commercial kitchens may exceed
only study to date to use hemoglobin adducts as a measure those of vehicles. Another important source of exposure is
of acrylamide exposure, this exposure was positively tobacco smoke. Furthermore, acrolein is produced endoge-
associated with estrogen-receptor-positive breast cancer, nously, especially in situations of oxidative stress. The EPA
though not with estrogen-receptor-negative breast cancer estimates that human exposure stems mostly from the
or breast cancer overall in a case–control study within a atmosphere, but set an oral Rfd of 5×10−4 mg/kg per day.
large prospective cohort study in Denmark [93].
As previously mentioned, glycidamide rather than Furan Furan (not to be confused with the PCDFs some-
acrylamide itself is thought to be responsible for times referred to as “furans”) is another chemical that arises
acrylamide-induced carcinogenesis in rodents. It was from heat treatment of certain foods, mostly in brewed
recently shown that when humans and rodents received coffee, but also detected in chili, cereals, salty snacks, soups
similar doses of acrylamide (corresponding to the mean containing meats, and a variety of other items. The USFDA
daily intake in humans), only ~9% of the relevant estimates mean intakes to be 0.26µg/kg bw in the general
metabolites excreted in urine were derived from glycida- population, the 90th percentile of intake being 0.61µg/kg
mide in humans, whereas they constituted at least 45% of bw per day. Infants are estimated to ingest a mean of 0.41
the determined metabolites in rats [89]. If glycidamide is µg/kg bw per day, with the 90th percentile being 0.99µg/kg
the proximate carcinogen of acrylamide, these results bw per day. A European analysis of 273 baby food items
suggest that humans are at considerably lower risk of indicated furan concentrations from below the detection
acrylamide-induced carcinogenesis due to this greatly limit to 112µg/kg. Assuming a daily consumption of 234 g
reduced ability for acrylamide bioactivation. Note, howev- of baby food, this would result in exposures ranging from
er, that there appears to be considerable interindividual <0.03 to 3.5µg/kg bw per day in a 6-month-old baby.
variation in the ratio of glycidamide mercapturic acid: Furan is a small volatile compound that can pass through
acrylamide mercapturic acid excretion, as indicated by the cell membranes and is easily absorbed after dietary
finding that this ratio ranged from 0.004 to 1.4 with a ingestion or inhalation and most likely also through the
median of 0.3 in 91 nonsmoking individuals with back- skin. It is metabolized by cytochrome P450 (CYP2E1) in
ground levels of acrylamide exposure [90]. Of particular the liver, the primary metabolite being carbon dioxide,
note, this ratio was found to be significantly higher in whereas the key cytotoxic metabolite is cis-2-butene-1,4-
children aged 6–10 years than in adults, and they also had a dial, a reactive dialdehyde. Effective first pass metabolism
significantly higher ratio of glycidamide hemoglobin and subsequent excretion are thought to largely prevent
adducts to acrylamide hemoglobin adducts. furan from being distributed to other tissues, but after
Recently, physiologically based pharmacokinetic/phar- administration of radiolabeled furan, about 15% of a single
macodynamic modeling based on hemoglobin adduct dose is detected in tissues, mostly in liver, lower amounts in
formation in conjunction with probabilistic estimate of kidney, still less in large intestine, and some in small
dietary exposure indicated that the lifetime excess cancer intestine, stomach, blood, and lung. Within 24 h after
risks for mammary, mesothelioma, thyroid, central nervous administration, about 80% of the dose is eliminated via
system, and thyroid tumors were in the range of 1–4×10−4 lung, urine, and feces, and radiolabel becomes essentially
[94]. In addition, it is known from occupational exposure undetectable 1 week after a single dose. After repeat
administration, furan derivatives accumulate mostly in the tubing, blood storage bags, food wraps, and components of
liver and kidney. paper and paperboard. In addition, phthalates are used as
The main reactive metabolite of furan, cis-2-butene-1,4- fixatives, detergents, lubrication oils, and solvents, in
dial, binds to proteins and nucleosides and is mutagenic and cosmetics and personal care products, and in medications.
genotoxic in vitro, whereas furan itself is only genotoxic. Since phthalates are not covalently bound to plastic and
Orally administered furan is clearly carcinogenic in rats, PVC-based products, they leach and vaporize from these
resulting in dose-dependent increases in hepatocellular ade- materials over time. As a result, these chemicals are
nomas and carcinomas as well as cholangiocarcinomas. In ubiquitous, and contamination of laboratory equipment
rats, it also dose-dependently induces mononuclear leukemia. makes it difficult to obtain reliable measurements of
A number of alkyl furans, dihydrofurans, furanones, and phthalate concentrations in food, other consumer products,
tetrahydrofurans can occur in food, but their metabolism is air, and dust. Determination of phthalate metabolites in
thought or has been shown not to yield reactive dialdehydes urine has become the method of choice for assessing total
similar to cis-2-butene-1,4-dial. They are therefore deemed phthalate exposure, but this makes it impossible to establish
unlikely to exhibit similar toxicities as furan. The oral Rfd what route and what products make the major contribution
established by the USEPA is 0.001 mg/kg bw per day. to total exposure. In addition, extrapolation of daily
phthalate intake from urinary excretion of their metabolites
Chloropropanols The Chloropropanols, 3-chloropropane- is hampered by several factors. Urinary phthalate metabo-
1,2-diol (3-MCPD) and 1,3-dichloropropanol (1,3-DCP), lite levels show high day-to-day and month-to-month
were first detected in acid-hydrolyzed vegetable protein, variability. Depending on the phthalate metabolite, up to
which is frequently used in soups, gravy mixes, stock cubes, four samples obtained 1–3 months apart may be necessary
and savory snacks. These chemicals are a by-product of the to classify exposure with a reliability of 80%. Furthermore,
production process of hydrolyzed vegetable proteins, which data on the fractional excretion of urinary phthalate
involves hydrochloric acid treatment at high temperatures, metabolites are extremely limited, and the use of different
resulting in the formation of chloropropanols from fats and published fractional excretion factors can result in up to
oils. In addition, 3-MCPD has been detected in soy sauce and tenfold differences in the median daily exposure estimate of
a variety of other foods, including toasted bread, grilled specific phthalates.
cheese, and fried batters. Their formation in these foods is not Phthalate exposure can occur via ingestion, inhalation,
well understood. and dermal absorption. What limited data are available on
Recently, the EU asked member states to assess the the subject suggest that dietary intake generally represents
dietary exposure to 3-MCPD of their inhabitants. This the major route of exposure [95], with fatty foods, such as
revealed that consumption from soy sauce alone ranged dairy, fish, meat, and oils containing the highest levels.
between 0.108 and 1.08µg/kg bw in different countries, and Inhalation may contribute considerably to exposure to
in some states, soy sauce contributed 70% of total 3-MCPD certain phthalates, such as di-n-butyl (DBP), benzyl butyl
intake. In other member states, sauces were the main source (BBP), and diethyl phthalate (DEP) [96]. Estimates of daily
of 3-MCPD exposure, and in Denmark, bread constituted phthalate intake derived from biomonitoring studies are
one third of total dietary intake. 3-MCPD is carcinogenic in summarized in Table 6, and the tolerable intake values from
rats at high doses, but not via genotoxic mechanisms. The various regulatory agencies are provided in Table 7. Note
EU set a TDI for 3-MCPD of 2µg/kg bw per day. In that German children were recently found to exceed the
contrast, 1,3-DCP is a genotoxic carcinogen for which the TDI set by the EFSA for DBP, whereas none exceeded the
FAO/WHO recommends keeping the levels in food as low TDI of 500µg/kg per day or the USEPA Rfd of 200µg/kg
as technologically achievable. per day for BBP [97].
Phthalates appear to be readily absorbed after gut lipases
and esterases hydrolyze them into their more easily
Contaminants arising from the packaging or packaging absorbable monoesters. The relatively polar and low
process molecular weight phthalates are conjugated to glucuronides,
whereas a large proportion of the monoesters of phthalates
Phthalates with higher molecular weights, such as di-(2-ethylhexyl)
phthalate (DEHP), di-n-octyl phthalate, and di-isononyl
Phthalates are dialkyl or alkyl aryl esters of 1,2-benzene- phthalate (DINP) undergo rather extensive oxidation before
dicarboxylic acid. They are used as plasticizers in plastic glucuronidation. Despite their lipophilic nature, phthalates
and PVC products, which have a wide range of applica- are metabolized and excreted via urine and, to a lesser
tions, including floor tiles, vinyl upholstery, toys, dispos- extent via the feces, within 3 days; consequently, bioaccu-
able medical examination and surgical gloves, medical mulation is not thought to occur.
Table 6 Estimated median or geometric mean (95th percentile) daily phthalate intake in microgram per kilogram body weight per day
Phthalate US, NHANES Germany, general Germany, children Germany, Germany, duplicate
[212] population [213] (2–14years) [97] students [214] diet [215]
DEHP 0.60 (3.05) 10.3 (38.3) 2.4 (5.7) 2.43 (3.95)

DEP 12.34 (93.33) 2.32 (22.1) 0.11 (0.18)
DBP 1.56 (6.87) 5.22 (16.2) 4.07 (14.9) 1.9 (5.3) 0.26 (1.35)
BBP 0.73 (3.34) 0.60 (2.52) 0.42 (2.57) 0.22 (0.910) 0.23 (0.38)
Di-n-octyl phthalate < LOD (0.42)
DINP 0.21 (1.08) 0.4 (1.5)
Reproductive toxicity It is extensively documented that in Insl3 mRNA and protein production. Effects on
certain phthalates exhibit toxicities in the male reproductive reproductive parameters have also been observed in rats
system of rats, mice, rabbits, and to a much lesser degree in following administration during prepuberty, while adult
hamsters. In particular, gestational exposure to DEHP, BBP, exposure results in adverse effects on the male reproductive
DBP, dipentyl phthalate (DPP), and, with far lesser potency, system only at very high doses. Interestingly, testosterone
DINP induces defects of the male reproductive organs. production may be affected differentially depending on the
These defects have been called the “phthalate syndrome,” exact timing of exposure.
which consists of hypospadias, cryptorchidism, gubernacu-
lar agenesis, testicular atrophy, underdeveloped or absent Human relevance The proximate developmental toxicants
epididymis, irreversible degeneration and atrophy of sem- of phthalates in rats and mice are thought to be their
iniferous tubules, reduced anogenital distance, and retained respective monoesters, although recent data suggest that
nipples/areolae. Many of these effects are attributable to the some of the secondary oxidative metabolites may exhibit
marked decreases in testicular and systemic testosterone even greater toxicity. Earlier data indicated that, at
production that are also observed in rats that were exposed equivalent doses, primates had lower serum concentration
to phthalates in utero and that constitute one of the most of mono(2-ethylhexyl) phthalate (MEHP, the monoester of
sensitive end points in the assessment of the male DEHP) than rats. From these data, it would be expected that
reproductive toxicities of phthalates. Other sensitive indi- humans would also be exposed to lower internal doses after
cators include decreases in sperm count and motility, and an oral DEHP intake compared to rats. However, this was not
increased incidence of morphologically abnormally sperm. confirmed in a study of a single human volunteer.
Of particular note, phthalates are unique in that they are the Comparison of the results obtained in this volunteer with
only group of endocrine disrupting chemicals investigated data from marmosets and rats suggests that human tissues
to date that causes agenesis of the gubernacular cords. The may actually be exposed to higher levels of MEHP.
development of this tissue depends on insulin-like hormone It has been proposed that the unconjugated monoesters are
3 (Insl3), a hormone that is produced by Leydig cells. It has the mediators of reproductive toxicity in rats since monoesters
been shown that gestational exposure to phthalates with undergo little glucuronidation in this species. In contrast, in
male reproductive toxicity results in pronounced reductions humans, the majority of phthalate monoesters and even the
secondary metabolites are present in the form of glucuronides
Table 7 TDI values for phthalates in microgram per kilogram body in urine and possibly in serum. However, there are indications
weight
that the percentage of free phthalate monoesters rises with
Phthalate SCF/EFSA EPA Health increasing exposure within the range found in the general
(oral Rfd) Canada population. In addition, there seems to be considerable
variability between subjects in the degree of conjugation. In
DEHP 50 20 44
conclusion, there is reason to believe that at least certain more
DBP 10 100a 63
highly exposed subgroups of the general population and/or
DEP 200 800 those with low capacity for glucuronidation may be at risk for
BBP 500b 200 adverse health effects from exposure to certain phthalates.
DMP (dimethyl 50 Accordingly, numerous regulatory agencies have affirmed
phthalate)
DINP 150 that the animal data on reproductive toxicity of certain
phthalates are likely to be relevant to humans.
a
300 are proposed, but not yet finalized Further confirmation comes from studies investigating
b
Increased from a previous TDI of 100µg/kg bw the correlation between phthalate exposure and a variety of
health outcomes. In adult men, an inverse association has resins, which have numerous applications, including poly-
been reported between exposure to certain phthalates, carbonate baby feeding bottles and other plastic storage and
measured as their urinary phthalate metabolite levels, and heating containers for food and beverages and epoxy lining
semen quality, such as sperm concentration and motility and of metal cans. It is an environmental pollutant that can be
frequency of DNA damage in sperm [98–100]. Changes in detected in water, including drinking water, soil, house dust,
reproductive hormone levels in association with urinary and residential air. However, the limited data available on
phthalate metabolite levels have also been observed, but the aggregate exposure estimates suggest that the vast majority
results are inconsistent, and the associations with several of human exposure occurs via oral intake of foods
hormones exhibited unexpected patterns and directions contaminated with bisphenol A leaching from plastic food
[101,102]. In another study, maternal urinary concentrations and beverage containers [107]. Note, however, that there are
of four phthalates metabolites (monoesters of DEP, BBP, indications that the bisphenol A concentrations in human
DBP, and di-isobutyl phthalate) during pregnancy were blood are much higher than would be expected from what is
inversely associated with anogenital distance in their male known about rodent and human metabolism and clearance of
children examined at the age of 2–36 months [103]. this chemical. This suggests that not all sources and routes of
Somewhat surprisingly, another group of researchers human exposure have been fully accounted for [107].
observed a significant inverse association between amniotic There is enormous variability in the estimates of
fluid phthalate metabolite levels (monobutyl) and anogen- bisphenol A exposure from food alone based on food
ital distance only in female, but not in male, neonates [104]. intake data combined with values measured in certain types
In addition, increased urinary phthalate metabolite concen- of food. Data collected by the USFDA indicate an intake of
trations have been associated with decreases in the thyroid 0.183µg/kg bw per day in US adults, which is lower than
hormone thyroxine, which prompted an investigation of a estimates reported from the EU (0.37 to 1.4), Japan (0.002
possible association between maternal phthalate exposure to 0.43), or New Zealand (4.8µg/kg bw per day). Exposures
during pregnancy and behavioral outcomes in neonates of infants and children are generally much higher, with the
[105]. Prenatal phthalate exposure was significantly asso- USFDA estimate for infants being 1.75µg/kg bw per day,
ciated with decreased quality of alertness and orientation similar to the 1.6µg/kg bw per day reported from the EU,
scores in female neonates only. whereas older infants and children aged 4–6 years were
Note that almost all of these studies used single estimated to ingest 0.8 and 1.2 µg/kg bw per day,
measurements of urinary phthalate metabolite levels. Given respectively. Estimates based on urinary excretion of
the relatively high day-to-day variability of this parameter, bisphenol A after hydrolysis of conjugates suggest consid-
a single spot urine sample is unlikely to accurately reflect erably lower daily intakes of 0.047µg/kg bw per day for the
chronic phthalate exposure. This may account for the US population overall, 0.065 in children aged 6–11 years,
unexpected patterns and directions of some of the reported and 0.71 in adolescents [108].
associations. At the same time, it raises some doubts The bioavailability of bisphenol A is much lower after
concerning the validity of the other observed correlations. oral ingestion than after intravenous or subcutaneous
Nonetheless, a somewhat more reliable indication that administration in rodents and primates. The concentrations
phthalates can affect human sex hormone production comes of unconjugated (free) bisphenol A measured in human
from a study that investigated possible associations between tissues and fluids range from 0.3 to 4.4 ng/ml even
six phthalate monoester concentrations in mothers’ breast excluding studies that used enzyme-linked immunosorbent
milk collected as additive aliquots during months 1–3 assay (ELISA; which is considered to yield overestimates
postnatally and serum concentrations of reproductive due to unspecific binding) [107,109]. Of particular note,
hormones in their male children [106]. The observed this chemical can be detected in a vast majority of fetal cord
correlation of certain monoesters with sex hormone- blood samples at rather high levels (up to 4 ng/ml in studies
binding globulin and with the ratio of luteinizing hormone using chromatographic methods). Free bisphenol A con-
to free testosterone suggested that neonatal phthalate centrations in human breast milk of 0.61 to 1.3 ng/ml have
monoester exposure via breast milk has subtle but signif- been reported, with total bisphenol A levels in these and
icant and dose-dependent effects on the levels of reproduc- other analyses between 0.46 and 3.41 ng/ml. Note that
tive hormones in male infants. some of the variability may be due to differences in the
analytical methods.
In humans, other primates, and rodents, there is
Bisphenol A extensive first-pass metabolism of bisphenol A yielding
mostly glucuronide conjugates with a minor amount of
Bisphenol A is a high-volume chemical used as the sulfate conjugates and little free parent compound remain-
monomer to synthesize polycarbonate plastic and epoxy ing in the circulation. In rodents, there is extensive
enterohepatic recirculation resulting in prolonged exposure reflecting differences in the route, timing, and duration of
to bisphenol A, including the free form. In contrast, humans exposure. Of particular note, dietary bisphenol A was found
and other primates do not show enterohepatic recirculation to decrease IFN-γ synthesis of mitogen-stimulated splenic
but instead rapidly excrete bisphenol A metabolites in urine. mononuclear cells in association with reduced IgG2a
Some regulatory agencies consider this to indicate that adult production and partial protection from glomerulonephritis
exposure to the low levels of bisphenol A in foods is not a in the NZB/WF1 model of lupus.
major concern. However, there are indications that human Several governmental or supragovernmental agencies
serum concentrations of free bisphenol A are in the same have commissioned risk assessments of bisphenol A,
range as those measured in rats after acute dosing [107]. In including the USA (CERHR Expert Panel for the National
addition, the enzymes involved in the detoxification of Toxicology Program (NTP)/National Institutes of Health
bisphenol A are not fully developed in the fetus and neonate (NIH)/Department of Health and Human Services), the EU
so that exposure in this age group may result in higher (EFSA), and Japan. In addition, the National Institutes of
levels of free parent compound and increased toxicity. Health/Department of Health and Human Services together
with the USEPA recently sponsored a meeting on bisphenol
Toxicities Bisphenol A is a selective estrogen receptor A health risk assessment. The conclusions largely depended
modulator that is several orders of magnitude weaker than on the interpretation of the available data on pharmacoki-
the endogenous estrogen 17β-estradiol in some outcomes netics in laboratory animals and humans and on the
but shows equal efficacy and potency in others. Earlier numerous low-dose studies conducted in recent years.
studies with bisphenol A at high doses indicated the lowest The EFSA concluded that the low-dose effects reported
observed adverse effect level of 50 mg/kg per day. in some studies were not robust and reproducible. It further
However, there are now >100 studies in rodents on the decided that recent data on major species differences
neuroendocrine and reproductive toxicity of prenatal, between rodents and humans in the metabolism of
postnatal, and adult exposure to lower doses of this bisphenol A justified reducing the uncertainty factor from
compound [110]. “Low dose” refers to doses within the 500 to 100 and therefore of establishing a fivefold higher
range of typical (nonoccupational) human exposures or full TDI of 0.05 mg/kg per day instead of the previous
doses below the current USEPA oral Rfd of 50µg/kg bw temporary TDI of 10µg/kg bw per day. The NTP concluded
per day. The results of these studies indicate that gestational that there was minimal to negligible concern over adult
and lactational exposure to bisphenol A, including via the exposures but expressed some concern over possible
oral route, can result in a number of organizational effects. neurological, behavioral, and prostate gland toxicities of
The term “organizational effect” refers to persistent alter- bisphenol A in fetuses, infants, and children.
ations of an organ or system that result from exposure In marked contrast, the participants of the meeting
solely during organ development. The disturbances arising sponsored by the NIH and USEPA deemed the evidence
from prenatal or perinatal bisphenol A exposure include convincing that the mean concentrations of unconjugated
alterations in brain structure and chemistry that are bisphenol A in humans correspond to the circulating levels
associated with impaired learning, mating, and maternal of bisphenol A seen in rats after acute low-dose oral
behavior. There is also extensive evidence that gestational exposure. They expressed the same level of confidence in
exposure to low-dose bisphenol A affects the male data showing that concentrations in the fetal mouse after a
reproductive organs, decreasing testicular testosterone maternal dose that produced adverse effects in numerous
levels, the weights of epididymis and seminal vesicles, experiments (25µg/kg bw) were within the range of
and daily sperm production but increasing the weights of unconjugated bisphenol A concentrations in human fetal
prostate and preputial glands. Decreased testosterone and blood. They further felt confident that the currently
sperm production have also been observed in juvenile and available data provide evidence for organizational changes
adult rodents treated with bisphenol A. In addition, there in the prostate, breast, testis, mammary gland, brain
are some studies showing effects of gestational bisphenol A structure, and chemistry and behavior after prenatal and/or
exposure on the female reproductive tract, in particular neonatal exposure and for substantial neurobehavioral and
early onset of puberty. More limited and at times reproductive effects after adult exposure in laboratory
controversial data suggest that bisphenol A can affect a animals. The experts also deemed certain other effects of
variety of immune parameters. Specifically, this chemical bisphenol A “likely but requiring confirmation.” These
was found to increase antigen-specific interleukin (IL)-4 included the reported effects of adult exposure to low-dose
and IL-10 production in animals primed with a variety of bisphenol A on the immune system, brain, and female
antigens and to also increase serum concentrations of reproductive system [110]. Whether and how these findings
antigen-specific IgE. Results on other cytokine and anti- should be used in setting a TDI for bisphenol A was not
body responses were somewhat more conflicting, possibly addressed at this meeting.
Semicarbazide eliciting IgE-mediated allergic responses, but approximate-

ly 90% of food allergies are due to eight foods: cow’s milk,
Azodicarbonamide is used in the manufacture of foamed hen’s egg, peanut, soy, wheat, tree nuts, shellfish, and fish.
plastic seals in the metal lids of glass jars and is used as a However, the most commonly implicated foods will vary
blowing agent in other food packaging materials. This by geographic region based on dietary preferences and even
chemical partially degrades during heat processing to form pollen counts, since some food allergy to fruits, vegetables,
semicarbazide. In addition, semicarbazide is a known or nuts is due to IgE antibodies cross-reacting with pollen
metabolite of the illegal veterinary drug nitrofurazone and allergens, with the pollen as the primary sensitizer. For
has been detected in seaweed derived products (e.g., example, in Switzerland, hazelnut allergy is the most
carrageenan), but these sources are unlikely to make major common food allergy in adults and is due to cross-
contributions to dietary exposure. Semicarbazide is detect- reacting IgE to a birch pollen pathogenesis-related protein
able in essentially all foods sold in glass jars, but baby [113]. In Israel, where sesame is incorporated prominently
foods were found to contain the highest concentrations (up in the diet, sesame seed is the most common cause of
to 54µg/kg). The dietary exposure from baby food was anaphylaxis in young children, and peanut allergy is rare
estimated to be 0.5µg/kg bw per day for 0–12-month-old [114]. In Singapore, edible bird’s nest soup is commonly
babies in the USA; 0.4 and 0.66µg/kg bw per day for implicated in pediatric food allergy, while in Japan,
Canadian babies aged 1–3 months and 6–9 months, buckwheat is an important cause of food allergy in
respectively; and between 0.23 and 0.53µg/kg bw per day school-age children [115,116]. Thus, although there are
for European babies [111]. In laboratory animals, semi- some regional differences, internationally, the Codex
carbazide exposure in utero or postnatally inhibits bone Alimentarius Commission recommended in 1999 that
mineralization and leads to skeletal deformities. In addition, member countries adopt the list of eight common foods
it is weakly carcinogenic. Various regulatory agencies, and take steps to ensure that manufacturers within member
including the USFDA, Health Canada, and WHO, do not nations list these foods or ingredients derived from these
consider the levels of dietary exposure as a human health foods on labels [117]. These eight foods were the focus of
threat since they are three to four orders of magnitude lower subsequent US legislation, known as the Food Allergen
than the NOAEL in animal studies. Nonetheless, since baby Labeling and Consumer Protection Act [118], that took
foods from glass jars may lead to high exposures in infants, effect in 2006. Under this legislation, all products contain-
who eat more food per unit body weight, it is strongly ing “Big Eight” foods require clear, “plain English”
recommended that azodicarbonamide be replaced by other labeling designed to help consumers navigate the confusing
suitable substances. The EU banned the use of azodicarbo- world of ingredient lists. For example, “non-dairy soy
namide in the sealing of food jars until more is known cheese” made from soybean, but also containing added
about the toxicology of semicarbazide. casein, was previously allowed to list “casein” as an
ingredient without disclosing that this is a cow’s milk
protein. To a parent of a child diagnosed with severe cow’s
Food allergens milk allergy, this might have appeared an excellent
substitute for regular cheese—with horrific consequences.
Food allergies are adverse immunologic reactions to The European Union, Australia, New Zealand, Canada, and
otherwise harmless food constituents, almost always pro- Japan have additionally published labeling guidelines
teins, and are most commonly IgE-mediated but also designed to ensure the safety of the food-allergic consumer
include cell-mediated delayed hypersensitivity responses, [119–122]. Table 8 lists the requirements in the different
most frequently gluten-sensitive enteropathy that affects countries or economic blocks.
just under 1% of the population (celiac disease). The Exceptions to the US labeling rule are allowed by a
prevalence of food allergies, excluding celiac disease, has petition and notification processes, similar to exceptions
been estimated to be 5–8% in children under the age of approved within the evolving framework of the recent EU
4 years and 3–4% in adults [112]. Directive (2003/89/EC) on allergen labeling that went into
For those affected by severe, life-threatening food full effect in November 2005 [121]. For example, in the
allergies in which even a trace amount of a food may EU, by a provisional exemption, casein, fish gelatin, and
trigger an anaphylactic reaction, food allergy is a significant fish isinglass (a product derived from fish collagen) will
food safety issue. Approximately 150 people are believed continue to be used as clarifying agents in beer, cider, or
to die from food allergy each year in the USA, but accurate wine without labeling since there is no evidence that such
numbers are not available. Food allergy is the most products pose a risk, while further research is performed. It
common cause of emergency room visits for anaphylaxis. would be quite confusing for consumers to read a wine
A wide variety of foods have been shown capable of label and find that it contains fish or cow’s milk after
Table 8 International food al-

lergen labeling requirements Country/block USA European Union Australia–New Zealand Canada Japan
[119–121]
Cow’s milk Yes Yes Yes Yes Yes
Hen’s egg Yes Yes Yes Yes Yes
Wheat Yes Yes Yes Yes Yes
Soy Yes Yes Yes Yes No
Peanut Yes Yes Yes Yes Yes
Tree nuts Yes Yes Yes Yes No
Fish Yes Yes Yes Yes No
Crustaceans Yes Yes Yes Yes No
Molluscs No No Yes Yes No
Sesame seed No Yes Yes Yes No
Mustard seed No Yes No No No
Celery No Yes No No No
Buckwheat No No No No Yes
consuming the product with no adverse reaction for years. manufacturers use provisional labeling, such as “may
A full exemption will be granted when the industry has contain peanuts” or “processed in a plant that also processes
satisfactorily demonstrated to a scientific panel that no peanuts,” but this is confusing for consumers and should be
allergen capable of eliciting an allergic reaction is present in avoided. Risk assessment as derived from toxicology
the finished products [123]; the literature in this area is practice is also applied to food allergy, and NOAELs and
growing [124–126]. LOAELs are being reported for food proteins involved in
Although the “Big Eight” foods account for about 90% IgE-mediated food allergy and for gluten in celiac disease,
of clinical reactions in children with atopic dermatitis in where there is more data. For example, in the EU, by 2012,
referral centers in the USA, the pattern of foods causing only foods with gluten levels by ELISA less than 20 ppm
reactions prompting emergency department visits is some- may carry a “gluten-free” label [129,130].
what different. In a recent multicenter study of emergency
department visits for food allergy, which gives a national
perspective, peanuts and tree nuts accounted for 21% of Genetically modified foods
visits, crustaceans 19%, and fish 10%, but fruits as a group
were significant and represented 12% of allergic reactions Genetically modified (GM) foods are foods made from
[127]. However, the “Big Eight” remains highly useful for plants or animals that have been given specific traits by the
labeling rules because the vast majority of individuals with insertion of one or more modified gene(s) or gene(s) from
food allergy will be helped, and specific fruits or vegetables another organism using the technique of genetic engineering.
are easier to avoid once the allergy is identified, and the Although genetically engineered animals also belong in this
patient receives appropriate education on avoidance. Fruits category and the USFDA is in the process of developing a
and vegetables are not as commonly found as “hidden” guidance on regulations of foods derived from such animals,
ingredients in processed foods but rather pose problems in the focus here will be on GM food crops.
restaurants or when dining away from home. On the other GM crops have gained rapid and widespread acceptance
hand, edible seeds (e.g., sesame, mustard, coriander, in the farming communities around the world, with the area
sunflower), which are sometimes associated with potential- dedicated to the cultivation of such crops increasing from
ly life-threatening allergies, are often found in trace 1.7 million hectares in six countries in 1996 to 125 million
amounts in a food and do not require disclosure in the hectares in 25 countries in 2008. Nonetheless, the USA and
USA, but sesame and mustard must be declared in the EU Argentina account for two thirds of all commercial GM crop
[114,128]. It would be prudent for food manufacturers to plantings and only five countries (USA, Argentina, Brazil,
list such ingredients, even if not required by a law, for the India, and Canada) account for 85% of them. Consumer
public good. acceptance of GM foods has not been equally enthusiastic,
At the current time, the focus is on labeling food particularly in many European countries because of fears that
allergens and maintaining good manufacturing practices to these foods represent risks for human health and the
avoid cross-contact and contamination of unlabeled food environment that are difficult to foresee accurately.
with an allergen. Such cross-contact can result in expensive The approach of the USFDA to regulating GM foods is
food recalls or even severe food allergic reactions. Some product-oriented, not focused on the process by which the
product was created. It is based on the concept that GM functions of the introduced trait and (2) possible unintended
organisms are not fundamentally different from nonmodified effects resulting from the insertion of the transgene into the
ones and that existing laws at the time of their first plant genome. Unintended effects can arise because it is
introduction were for the most part adequate for regulation impossible to predict where in the genome a transgene
of GM products. Except for a comprehensive scientific review becomes inserted, how many copies are inserted, and
of a GM tomato in 1992, the USFDA has reviewed GM foods whether the transgene is rearranged in the process. Depend-
via a voluntary consultation process during which the ing on the place of insertion, the introduced gene can alter
developer of the GM food presents data on the modification metabolic pathways or induce the expression of previously
and the possible allergenicity and toxicity of the expressed silent genes, possibly resulting in increased expression of
product, the composition and nutrient profile, and, in some toxins, antinutrients, or allergens or reduced levels of
cases, the results of feeding studies comparing the GM food to essential nutrients. Therefore, key components of the
its nonmodified counterpart. Although in 2001 the USFDA comparison of GM foods to their mostly isogenic counter-
proposed a rule to make this process mandatory, we have been parts are compositional, molecular, phenotypic, and agro-
unable to find a finalized rule on this issue. nomic analyses. The compositional analysis focuses on
It seems probable that a majority of consumers are macronutrients and micronutrients, antinutrients, and tox-
unaware to what extent “conventional breeding” of plants icants. In order to compare the composition of GM foods to
relies on extensive manipulation of the plant genome. This their traditional counterparts, it is absolutely essential to
includes techniques that have been in commercial use since have information on the natural ranges of variation of the
the 1920s, such as interspecific and intergeneric hybrids key components that should be analyzed. Remarkably, it
and accelerated mutagenesis (i.e., the use of chemical and took almost 10 years to develop consensus documents on
radiation treatments to induce mutations and chromosome the natural composition and its variability of important GM
rearrangements), to mention but a few [131]. This raises the crops, such as maize, soybean, canola, potato, sugar beet,
question of whether risk assessment should be confined to and bread wheat [132]. Others are still being developed.
GM foods. Interestingly, Canada appears to be the only Whether further data are called for before a GM product
country where regulatory oversight is the same for all new can be marketed largely depends on whether or not the
agricultural commodities, regardless of the process by molecular and compositional analysis demonstrates sub-
which they are created (www.agbios.com/static/cscontent/ stantial equivalence and is decided on a case-by-case basis.
REGCanada-USAEN_printer.html). GM foods are sub- Generally, it is only if the results of these analyses provide
sumed under the category of novel foods, i.e., products indications that unintended effects have occurred (e.g., that
that do not have a history of safe use as a food or foods that the composition is modified substantially) that animal
are manufactured in ways that significantly change their feeding trials may be required. In such cases, most
properties. The category “plants with novel traits” encom- regulatory agencies require only 90-day feeding trials in a
passes plants that are neither familiar nor demonstrate single species. Such trials have revealed relatively subtle
substantial equivalence to established plants, regardless of but significant effects on growth/weight gain, hematologi-
whether they arise from recombinant DNA techniques or are cal and immunological parameters, and organ histopathol-
produced by chemical mutagenesis, cell fusion, and even ogy with numerous GM foods [133]. These significant
conventional crossbreeding. The safety assessment is based differences compared to the included control groups are
on a comparison of the new food to a traditional counterpart. often dismissed as being within the normal range for that
In all other countries, it is not the product but the process species, raising the question of why anybody bothers to
of genetically modifying a plant that triggers regulatory include a control group at all.
oversight. However, the concept of “substantial equiva-
lence” also forms the cornerstone of the safety assessment Allergenicity assessment of GM foods
of GM foods required for their authorization. This concept
was first introduced by the OECD and has since been The safety evaluation of GM foods contains a unique
adopted and elaborated by various international agencies, feature: it requires assessment of the potential allergenicity
such as the FAO/WHO, the International Life Sciences of the transgenic protein(s) and possible alterations in the
Institute, and others. It means that a comparative approach allergenicity of the whole food. Increased allergenicity can
is applied in the risk assessment of GM foods, which is not result when (1) the protein encoded by the introduced gene
intended to guarantee absolute safety but to ensure the same is identical or cross-reactive with an existing allergen; (2)
level of safety as provided by traditional foods. The safety expression of the transgene results in the de novo creation
assessment of GM foods usually follows a stepwise of a food allergen (either because the transgene encodes a
procedure that focuses on two main categories of potential novel protein not previously encountered in the human food
hazards: (1) those related to the intended properties and chain or because the transgenic protein is rendered
allergenic by alterations in the gene sequence or posttrans- Several expert bodies and regulatory agencies, including
lational modifications in the host), or (3) the introduction of the FAO/WHO, Codex Alimentarius Commission, USFDA,
the transgene increases the levels of—and exposure to—an and the European EFSA, have published guidelines for
endogenous antigen due to unintended effects on plant conducting the assessment of potential allergenicity of
metabolism. In addition, there are ongoing attempts to transgenic proteins. They all recommend a step-by-step
apply genetic engineering to the elimination of allergens decision tree process that uses a weight-of-evidence
from staple foods such as potatoes, rice, tomatoes, and approach (see Fig. 1 for the FAO/WHO decision tree). It
apples or highly nutritious foods such as peanuts [134]. includes consideration of whether the source of the gene to
A very limited number of items accounts for most food be transferred is known to be associated with allergic
allergies, with cow’s milk, egg, peanut, soy, wheat, and fish reactions. The FAO/WHO discourages the use of genes
representing the most common allergens in children and from common allergenic foods. Regardless of whether the
peanut, tree nuts, fish, and shellfish being the predominant source is a known allergen or not, the next step should be a
allergens in adults While allergy to cow’s milk, egg, soy, or sequence comparison between the transgenic protein(s) and
wheat can range in severity from mild to severe, most cases known allergens in order to detect possible significant
are mild and are outgrown by age 5, while allergies to sequence homologies suggestive of cross-reactivity.
peanuts, tree nuts, fish, and shellfish are usually persistent. According to the FAO/WHO guidelines, cross-reactivity
Only about 20% of children with peanut allergy will needs to be considered if there is (a) identity of six
outgrow it by age 5 to 6 [135]. Peanut or tree nut allergy contiguous amino acids or (b) more than 35% identity in
is often severe and together accounts for 90% of reported the amino acid sequence of the expressed protein over any
fatalities to foods in a US case series reported by the Food stretch of 80 amino acids. The first criterion is now
Allergy and Anaphylaxis Network [136,137]. This suggests considered to result in an unacceptably high number of
that there is a broad spectrum of allergenic potencies false positives [141], and common practice is to use identity
ranging from the inability to cause sensitization at one end of eight rather than six amino acids. The second approach is
to the capability to persistently sensitize and provoke severe also likely to yield very conservative estimates of potential
allergic reactions at the other end. Further evidence that all allergenicity, but whether applying more stringent criteria
proteins are not equally allergenic comes from the (e.g., 50% homology) would improve the prediction
observation that allergenic proteins share certain functional without unduly increasing the number of false negatives
and biochemical properties. Specifically, they belong to a remains to be established. It has been argued that the
small proportion of all known sequence-based (2%) or epitopes recognized by IgE are more likely to be confor-
structural protein families (5%) and most frequently exhibit mational and that the use of significant homology over
hydrolase, metal, or lipid binding and transport activity or larger stretches of the protein would yield more relevant
are involved in cytoskeleton organization and biogenesis information than identity over six or eight amino acids.
[138]. Food allergens in particular most often are members Note, however, that food allergens are generally believed to
of the prolamin, cupin, or cysteine protease superfamilies, be partially denatured or digested by the intestinal tract
and many of them belong to the seed storage or before coming into contact with the immune system,
pathogenesis-related proteins. In addition, allergenic food especially in the initial immune response As a result, IgE
proteins commonly, but not always, are abundant in their binding to linear epitopes is likely to be of much greater
respective foods, fall into the size range between 10 and importance in severe food allergy than recognition of
70 kDa, and are glycosylated, heat stable, and resistant to conformational epitopes [138]. Hence, some investigators
acid and enzymatic proteolysis. feel that global sequence similarity may not be suitable for
Nonetheless, predicting the allergenicity of novel (trans- predicting cross-reactivity among food allergens and favor
genic) proteins remains difficult since, to date, no unique sequence homology of linear epitopes for assessing
feature or set of features that confers allergenic properties to potential allergenicity. However, data are incomplete on
proteins has been identified. Note, however, that new the importance of conformational epitopes in different food
approaches are becoming available. There now is a allergens, in large part because their study has been
structural database of allergenic proteins (http://fermi. technically difficult to carry out [142]. Current guidelines
utmb.edu/SDAP/) that includes 3-D models of all cataloged also frequently recommend serum screening in the case that
allergens and provides a variety of analytical and compu- the sequence comparison of the transgenic protein with
tational tools that take some of the known features of known allergens did not reveal significant sequence
allergenic proteins into account [139,140]. The application homology. Specific serum screening refers to testing of
of these tools is thought to hold great promise for better whether serum IgE from individuals with documented
predicting the allergenic potential of proteins than the clinical food allergies to the source of the transgene
currently available approaches [141]. recognizes the novel protein (either because it is an as yet
Fig. 1 FAO/WHO decision tree Source of gene

for the assessment of the aller- yes allergic no
genic potential of foods derived
from biotechnology (www.fao.
org/ag/agn/food/pdf/allergygm. yes
pdf) Sequence homology Sequence homology
yes
no no
no
Specific serum screen Targeted serum screen
yes no
yes
Pepsin resistance
&
Animal models
Likely allergenic
+/+ +/– –/–

High Low
Probability of allergenicity
unknown allergen or is cross-reactive with a protein to threatening reactions. Therefore, currently available studies
which the patient produces IgE). Targeted serum screening on the allergenicity of novel proteins often complement
is considered useful either when specific serum screening serum screening with skin prick testing (SPT). The release
yields negative results or when the transgene is derived of histamine from the rat basophilic leukemia line 30/25,
from a source with unknown allergenic potential and transfected with the human IgE receptor, may represent a
encodes a protein product that does not show significant valid in vitro alternative [141].
sequence homology with known allergens. Targeted serum If sequence homology analysis and serum screening
screening refers to testing the protein of interest with sera have not yielded any indications of allergenicity, it is
from patients sensitive to substances that are broadly related recommended to examine the susceptibility of the trans-
to the source material of the gene. (The FAO/WHO genic protein to pepsin digestion. The rationale is that
distinguishes six source groups: yeast/molds, monocots, peptides large enough to be recognized by the immune
dicots, invertebrates, vertebrates, and “others”). system need to survive the digestive process in order for
Conducting serum screening in a manner that accurately allergic sensitization to occur. Consistent with this hypoth-
predicts allergenicity is considered to remain “extremely esis, a majority of food allergens associated with clinically
challenging” [141]. One of the major obstacles lies in severe reactions are resistant to pepsin degradation in vitro,
obtaining sufficient amounts of well-characterized specific even though there are examples of pepsin resistant food
donor sera. In addition, it is crucial to use an appropriately proteins that do not elicit food allergies.
validated IgE assay in which precautions are taken to Most guidelines for allergenicity assessment of isolated
prevent the much greater abundance of specific IgG food proteins (or whole foods) also recommend the use of
antibodies or the presence of clinically irrelevant cross- animal models but point out that no generally accepted and
reactive carbohydrate determinants from obscuring the validated models are currently available. An appropriate
results [141]. It also needs to be kept in mind that animal model should reproduce the major features of
sensitization (the presence of IgE) can commonly exist in human allergy, i.e., sensitization and provocation of allergic
the absence of clinical allergic symptoms. Ideally, double- symptoms, at physiologically relevant doses. The oral route
blind, placebo-controlled food challenges should be per- of exposure is thought to be most pertinent, although
formed in order to demonstrate biologically relevant humans may also develop food allergies due to cross-
allergenic activity. However, this may not be possible, reactivity with inhaled allergens or after transdermal
particularly if the novel protein exhibits similarity to a exposure. In fact, transdermal sensitization to peanut in
known allergen that is associated with severe or even life- murine models is extremely effective [143], and current
human prospective studies suggest that household environ- they specifically call for an assessment of the allergenicity
mental exposure to peanut, especially in children with atopic of the whole GM product (www.efsa.europa.eu/EFSA/
dermatitis, is a risk factor for peanut allergy while early oral efsa_locale-1178620753812_1178620775770.htm). How-
ingestion may be protective [144]. However, as in humans, ever, they provide essentially no details on how such an
animals generally develop oral tolerance to ingested proteins, assessment should be conducted except to say that the
necessitating the use of adjuvants when the test substance is allergen repertoire of the GM food should be compared to
given orally or by an alternate route of exposure. that of its non-GM counterpart using modern “profiling
The need to assess the allergenicity of GM foods has techniques.” We would also like to emphasize again that
provided a major impetus for the development of appropri- currently the assessment of potential allergenicity is only
ate animal models. Several mouse strains, including Balb/c, required for transgenic proteins (foods). However, more
A/J and C3H/HeJ, and the Brown Norwegian rat represent conventional breeding methods result in more pronounced
high IgE responder strains and thereby resemble the human changes in the expression of untargeted genes compared to
phenotype that is susceptible to developing IgE-mediated transgene insertion [145]. In addition, novel proteins
allergies. Other models include dogs and neonatal pigs, (resulting from novel open reading frames) that are present
which offer some advantages because of their larger size in certain conventional food crops show a similar number
but are thought to lend themselves more readily to of sequence homologies with known allergens and also of
mechanistic studies rather than routine safety assessments. identical stretches with indications that they are true
Recent efforts have focused mainly on mouse models epitopes as do transgenic foods [146].
because of the ready availability not only of inbred strains
but also of reagents for characterizing their immune Applications of the assessment of potential allergenicity
responses [141]. It is recommended to not only examine
specific IgE production (sensitization) but to also obtain a Relatively early in the commercial development of GM
readout of clinically relevant end points, such as allergic foods, there was an attempt to improve the nutritional
manifestations after challenge. In addition to sensitization, quality of soybeans, which are relatively deficient in the
the ability to escape the induction of oral tolerance is an essential amino acid methionine. For this purpose, a
important prerequisite for allergic responses, and its methionine-rich Brazil nut protein (2S albumin) was
assessment has been suggested to offer valuable insights inserted into the genome of a soybean variety. Even though
[141]. Of note, recent experimental data have provided the allergenicity of Brazil nuts was well known at the time,
evidence that pepsin stability is an important predictor of information on the major allergens in this food was still
sensitization but that oral tolerance induction requires lacking. Using a variety of techniques, including radioal-
resistance to digestion by both pepsin in the stomach and lergosorbent (RAST) assays, sodium dodecyl sulfate poly-
trypsin in the intestine. There are now several indications acrylamide gel electrophoresis and immunoblotting, and
that the food matrix can affect the allergenicity of SPT, it was clearly demonstrated that 2S albumin was
individual food constituents by altering their digestibility recognized by serum IgE from subjects with proven Brazil
and solubility. This raises the question of whether the use of nut allergies and probably represented a major Brazil nut
purified (and often microbially expressed) proteins repre- allergen [147]. Commercialization of this methionine-rich
sents the most appropriate approach to the allergenicity transgenic soybean was abandoned.
assessment of GM foods. Food matrix effects are but one of In most other published studies, serum screening was not
several issues that need to be clarified before any of the conducted during the premarket allergenicity assessment
currently available animal models can be validated. It but as a form of postmarketing surveillance. The methods
should be kept in mind that animal models by themselves included RAST inhibition, immunoblots, and/or ELISA on
cannot capture all traits of human food allergies and should sera of subjects sensitized to the non-GM food and, in some
not be expected to do so but may yield important hints as to cases, from potentially sensitive subjects with a variety of
possible allergenicity of GM (and more conventionally bred other food or inhalant allergies. In several instances, serum
novel) foods. However, in the context of risk/safety screening was combined with SPT or histamine release.
assessment, animal models always need to be used in These investigations provided no evidence that the allerge-
conjunction with other methods. nicity of transgenic crop plants, including soybean, maize,
Note that the guidelines concerning the allergenicity tomatoes, and potatoes, differed significantly from that of
assessment of GM foods generally are focused exclusively their conventional counterparts. There are, however, isolat-
on the transgenic protein(s), even though it is recognized ed examples of differential reactivity to wild-type and GM
that unintended changes in plant metabolism may result in foods and at least one case where SPT reactivity was
qualitative or quantitative changes in existing allergens. The confined to GM soybean, but these assays were done in
European EFSA guidelines are a notable exception in that individuals with sensitization to soy, not clinical allergy,
and the wild-type soy was not the parent cultivar of the GM transgenic protein that altered its immunogenicity. Specifi-
soy [148,149]. cally, the transgenic pre-pro-peptide underwent different
However, the well-publicized case of StarLink corn posttranslational processing (glycosylation and possibly
provides an example of the detection of a potential novel cleavage) in the pea compared to the bean. Intragastric
food allergen through the use of pepsin and trypsin administration of bean or nontransgenic pea seed meal did
digestion in conjunction with other considerations. This not provoke a specific IgG1 response even after intratracheal
transgenic corn was generated by inserting a gene derived or subcutaneous challenge with purified αAI. In contrast,
from Bacillus thuringiensis (Bt) that encodes an insecticidal oral administration of transgenic pea seed meal induced
toxin belonging to the crystal protein family, Cry9c. The immunoreactivity to αAI (increased αAI-specific IgG1
original sequence of this gene was modified to yield the levels) and failed to induce oral tolerance as assessed by
truncated active toxin rather than the bacterially encoded significant delayed-type hypersensitivity responses to foot-
protoxin that needs to be cleaved by trypsin into its active pad challenge and airway hyperreactivity and pulmonary
form. It was also modified to resist further trypsin digestion eosinophilia after intratracheal challenge with purified αAI.
to an inactive form by a single amino acid substitution that Importantly, extensive boiling of transgenic peas did not
eliminated the relevant trypsin cleavage site. Because Cry9c abrogate their ability to prime for pulmonary hyperreactivity,
was found to be resistant not only to trypsin but also to eosinophilia, and mucus secretion after intratracheal chal-
pepsin digestion and heat treatment, it was considered a lenge. Most notably, oral administration of transgenic pea
potential allergen even though it did not show any sequence seed meal also resulted in enhanced IgG1 responses to
homology with known allergens. As a result, the USEPA several other pea seed proteins. Furthermore, concomitant
approved StarLink corn for animal feed use only, but not oral administration of transgenic pea seed meal and
for human consumption [150]. In studies submitted by the ovalbumin (OVA) resulted in cross-priming, as evidenced
company at a later time point, specific serum screening by significant increases in OVA-specific IgG1 levels along
indicated that the allergenicity of StarLink corn did not with airway hyperreactivity and pulmonary eosinophilia
significantly differ from that of conventional corn. On the compared to OVA treatment alone (which does induce oral
other hand, some undigested Cry9c could be detected in the tolerance). Unfortunately, αAI-specific IgE was not deter-
blood of animals that were fed the pure protein. In addition, mined in this study. Although it has been suggested that
it was capable of inducing allergic reactions in Brown antigen-specific IgG1 represents a surrogate of IgE in the
Norway rats, but these results were not considered in the mouse [155], the two are regulated differentially, and in
evaluation by the USEPA because this animal model had particular antigen-induced airway hyperresponsiveness has
not (and has not) been validated for assessing the allergenic been shown to occur in animals with specific IgG1 responses
potential of transgenic proteins [151]. Unfortunately, in the absence of significant specific IgE production.
StarLink corn did get into the human food supply and 51 Therefore, it cannot be determined whether transgenic pea
people claimed allergy to it, but no clinical food allergy was meal induced classical IgE-mediated hypersensitivity. None-
ever confirmed [152,153]. theless, these results underscore that transgenic expression of
Of note, since it proved difficult to isolate the protein a nonallergic protein (endogenous αAI in beans) in a
from transgenic corn, the USEPA allowed the microbially different host crop can exhibit altered immunogenicity, most
produced trypsinized active toxin to be used as a test likely due to differential posttranslational modifications.
substance. This is problematic since the resulting Cry9c They further illustrate that such an altered transgenic protein
would not be glycosylated, whereas it might be subject to can enhance the immunogenicity of concomitantly adminis-
glycosylation in plants [150]. There are numerous indica- tered food antigens. This is an aspect that is not addressed at
tions that IgE binding to an allergen that is solely due to the all in the safety assessment of GM foods.
recognition of cross-reactive carbohydrate determinants is Whether GM foods are safe for humans, animals, and the
clinically irrelevant. However, in the case of specific environment cannot be determined from the existing data. It
allergens, it has been shown that the carbohydrate moieties has been calculated that the margin of safety between
of certain plant glycoproteins are required for IgE recogni- animal intake in 90-day feeding trials and human intake is
tion of allergenic epitopes and/or activation of helper T generally greater than 100 [133]. This might be reassuring
cells, whereas in other cases such carbohydrates can inhibit if one could have confidence in the quality of the studies on
specific T cell responses. Glycosylation may also influence which these calculations were based. It certainly does not
the thermostability and resistance to proteolysis of proteins. promote consumer confidence in the approval process or
A more recent study further underscores the potential the safety of GM foods to find out that the reanalysis of
importance of glycosylation [154]. It was shown that the data originally submitted for approval of MON863 corn
transgenic expression of a bean protein (α-amylase inhibitor- revealed indications of sex-dependent liver and kidney
1, αAI) in peas resulted in structural modifications of the toxicity in mice fed with this product for 90 days [156],
whereas the original publication of the results from this ex- techniques. There is no generally accepted definition of
periment considered the significant differences they found “nanomaterials.” The Scientific Committee on Emerging
not biologically meaningful. It is also not reassuring that and Newly Identified Health Risks, an advisory committee
most regulatory agencies require only 90-day feeding trials of the EU Commission, proposed the definition of “any
in a single species—and only in cases where a feeding trial is form of a material that is composed of discrete functional
indicated by the compositional and molecular analyses. parts, many of which have one or more dimensions of the
Many consumer protection groups in the USA and order of 100 nm or less.”
Europe demand more extensive safety testing of GM It has been estimated that close to 400 manufacturer-
products. As pointed out above, it may be more appropriate identified nanotechnology-based consumer products were
to take the Canadian approach and include not only GM on the market by 2007. In the food production chain,
foods but other novel foods derived from more conven- nanotechnology products are (claimed to be) used as
tional plant “breeding” techniques in such safety assess- nanosensors for the detection of contaminants, toxins, or
ments. Whether the “substantial equivalence” concept microorganisms; in water purification filters; pesticides;
proves tenable in the safety assessment of GM foods food packaging (either as nanosensors for the detection of
remains to be established. As Millstone et al. [157] pointed food deterioration, in nanosized silver sprays with antibac-
out, developers of GM foods want to have the best of all terial action, or for some packaging materials themselves);
possible worlds by arguing that their foods are sufficiently and also in certain functional foods or food supplements.
novel to justify patenting them, but not so novel as to Typically, the biological activity of particles increases
require risk assessment. They further underline that the with decreasing particle size. This is because there are a
“substantial equivalence” approach provides “an excuse for greater number of particles with a larger surface area per
not requiring biochemical or toxicological tests.” unit mass, resulting in increased potential for biological
Ironically, severe food allergy to an unregulated soy interactions. There are already indications that certain
substitute, lupin seed, has emerged in markets where there nanomaterials can generate reactive oxygen species, induce
is distrust over GM soy. Lupin, previously mainly used as oxidative stress, alter mitochondrial function, resulting in
an animal feed rather than human food, has been promoted apoptosis or other forms of cell death, interact with proteins
since about 1997 as an excellent protein source, high in in ways that alter their immunogenicity, damage DNA, and
fiber and able to replace egg and butter in some products. It interfere with cell cycle regulation [159].
can be added to breads and pastas instead of soy flour. Whereas the inhalation and, to a lesser extent, dermal
However, there is significant clinical cross-reactivity with routes of exposure to certain nanomaterials have been
peanut, and the first cases of anaphylaxis were reported in investigated to some extent, there is little information on the
1999. A recent study has shown that approximately 4% of fate of nanoparticles after ingestion [159]. What few data
children with peanut allergy are clinically allergic also to are available suggest that the uptake from the gastrointes-
lupin [158]. The 2S albumin seed storage proteins from tinal tract after oral administration may be quite efficient for
lupin have significant homology with the counterparts in some types of nanomaterials, whereas the bioavailability of
peanut. Thus, the potential for new food allergens exists not others may be very limited. A recent study investigated the
only for GM foods and foods derived from traditional plant toxicology of orally administered silver nanoparticles,
breeding that enhance desirable characteristics (and thus which are extensively used as antimicrobial films in food
might increase certain allergens) but also with unmodified wraps and a variety of other applications [160]. Dose-
foods newly introduced to a population. dependent accumulation of silver in all tissues examined
suggests at least some bioavailability after oral administra-
tion. Interestingly, significantly higher levels of silver were
Emerging food safety issues detected in the kidney of female compared to male rats at
all doses. For most other nanomaterials, information on
Nanomaterials their absorption, tissue distribution, metabolism, and excre-
tion is incomplete or absent.
It has been conjectured that the impact of nanotechnology Since the exposure to nanomaterials of the general
in the twenty-first century will surpass that of the industrial population is bound to increase dramatically in the recent
revolution during the nineteenth century. The prefix nano future, detailed knowledge of nanomaterial absorption,
refers to a measure of 10−9 units. Nanotechnology refers to distribution, metabolism, and excretion after oral and other
the design, production, and application of structures, routes of exposure and nanomaterial toxicology must
devices, and systems by manipulating shape and size at become the focus of intense research. Their impact on the
the atomic, molecular, or macromolecular scales. Nano- environment is another topic that urgently needs to be
materials can result from “top-down” or “bottom-up” addressed. Such research faces numerous challenges. The
physicochemical properties of nanomaterials may differ define the role of T cell reactivity to various insulin and
fundamentally from their macroscale counterparts, as may GAD65 peptides, but whether such observations reflect
their toxicological profile [161]. Therefore, the safety of a direct causation with food is still very much unclear [174–
macroscale compound does not provide any indication of 183]. Indeed, it is abundantly clear that further study of the
the potential toxicities of its nanoscale counterpart. Nano- mucosal immune system, and including Peyer’s patches, is
materials come in various shapes, including nanosheets, critical for dissecting mucosal tolerance [184–186]. Lastly,
nanorods, nanotubes, nanodots, nanowires, or nanofibers, we should note the role of the innate immune system,
each of these structure having its own individual character- including the potential of food and food additives to
istics. These include shape, size (surface area, size distribu- promote inflammation [187]; discussion of innate immune
tion), mass, chemical composition, surface structure, surface responses is beyond the scope of this review, but recent
chemistry, surface charge, porosity, solubility, and aggrega- literature is cited [188–195].
tion/agglomeration state. These characteristics may all influ-
ence the behavior, including the biological activity, of
nanoparticles and should therefore be described in any testing Mixture toxicology
of nanomaterials. There is uncertainty over whether mass,
particle number, or surface area is the most meaningful dose The current risk assessment paradigm—hazard identifica-
metric [162]. This currently needs to be evaluated on a case- tion, exposure assessment, and risk characterization—is
by-case basis, and it is recommended to express the dose generally applied to single chemicals via a single route of
response in terms of all three parameters whenever possible exposure. In order for this approach to be valid, each one of
in order to enable quantitative interpretation of the collected the multitude of natural and synthetic chemicals to which
data and allow them to be compared to other results. we are exposed would have to act independently of all the
Because of these difficulties, there is as yet little to no others. If confirmation was needed that we are indeed
information on the extent of our exposure to nanomaterials exposed to multiple chemicals everyday, it certainly has
or their potential hazards, the two fundamental require- been provided by studies showing the presence of metab-
ments for risk assessment. Several consumer protection olites of numerous phthalates in urine [196] or the
groups have called for a moratorium on all marketing of simultaneous detection of various PCB congeners, DDT,
nanomaterials until it has been shown that their production DDE, hexachlorocyclohexane, and hexachlorobenzene in
and use does not present a significant risk to human health serum of women who also excreted OP pesticide metabo-
and the environment. Many scientists consider this to be lites in their urine [58]. Furthermore, the focus on a single
excessive as long as all the relevant regulatory agencies route of exposures would only be justifiable if all other
evaluate whether existing regulations provide sufficient routes made negligible contributions, which does not
protection and stay alert to the possible requirement for new appear to be the case for numerous compounds.
regulations as new applications arise. In recent years, there has been increasing recognition
that information on the cumulative risk from multiple
Autoimmunity and food chemical exposures is urgently needed, and the field of
“mixture toxicology” has emerged. First, it needed to be
One subject that has not been discussed independently is determined what substances would constitute good candi-
the issue of foods in autoimmunity. We have discussed dates for mixture studies. The combined toxicological
select data on mercury and autoimmunity, and the purpose effects of mixtures of compounds can take one of three
of this review is not an exhaustive discussion of foods and, forms: independent action (also called response addition),
for example, inflammatory bowel disease. However, we dose addition, or interaction. Substances with diverse
would be remiss if we did not note that not only foods, i.e. modes of action will each exert their individual effects
milk, but also the possibility of food contaminants can elicit whether alone or present in combination with other
significant immune reactions [163]. These are exemplified substances, i.e., they will display independent action. Dose
by detection of antibodies against raw and processed foods, addition is commonly seen with substances acting via a
the role of gluten in celiac disease, the potential impact of common mode of action and represents a situation where
probiotics on the immune response, the developing data on one compound in the mixture can be replaced by another
vitamin D and immunity, and the influence of fatty diets one, provided it is present at an equally effective dose level.
and the immune response [167–173]. In addition, and The term interaction refers to mixture effects that are
perhaps of greater interest, is the potential role of food as an greater (synergistic, potentiating) or smaller (antagonistic)
initiator of autoimmune diabetes. This is an issue that has than predicted on the basis of dose addition. Once a group
attracted attention for several decades but still remains of compounds with a common mode of action has been
elusive. Recent literature on diabetes has continued to identified, it needs to be determined whether these
compounds exhibit dose additivity. Most commonly, this is the TEF approach should yield identical results, regardless
done by establishing dose response curves for each of the of the index compound used [197]. Therefore, the observed
individual compounds, then using these data to predict their dependence of the exposure estimates on the index
combined effects under the assumption of dose addition, compound reflects the incompleteness and uncertainty
and finally establishing whether there is agreement between associated with the database on which the TEF values were
the predicted and the observed results. based. In its cumulative risk assessment of OP pesticides,
There are several methods for conducting cumulative the USEPA assumed that the effects of these compounds
risk assessment for substances that have been shown to act would be characterized by dose addition. However, even
in a dose-additive manner [197]. One of these approaches, studies cited by the USEPA show that certain combinations
the use of toxic equivalency factors (TEFs), was originally of OP pesticides can have synergistic effects particularly in
developed by the USEPA for the toxicity of mixtures the low-dose range, although the synergism was of
consisting of dioxins and dioxin-like compounds. It has relatively small magnitude [200–202]. These synergistic
since been adopted and recently updated by the WHO [62], effects are likely to result from the ability of certain OP
and WHO TEFs are used widely for the assessment of pesticides to also inhibit carboxyl esterases, which are
human exposure to dioxins and related substances. It involved in OP pesticide detoxification.
expresses the potency of chemicals relative to that of an Together, these finding underscore that, although the
index compound (TCDD in the case of dioxins and dioxin- USEPA used tremendous amounts of resources to conduct
like compounds). The exposure to each of the chemicals in its cumulative risk assessment of certain groups of
the common mode of action group is then multiplied by the pesticides, the validity of the conclusions remains some-
TEF in order to express all exposures as a fraction or what doubtful. Despite these uncertainties, however, a
multiple of the index compound. The resulting values are cumulative risk assessment approach is certainly preferable
summed to provide the total exposure in TEQs. to the single chemical approach and has already resulted in
the revocation of 3,200 tolerances and the modification of
Applications In 1996, the US Congress passed the Food another 1,200 by the USEPA.
Quality Protection Act (FQPA), which requires the USEPA, Several other types of pesticides are classic antiandro-
which is responsible for setting tolerances for pesticide gens in that they bind to the AR and thereby inhibit
chemical residues in food, to assess the cumulative human androgen binding and induce malformations in the devel-
health risk from simultaneous exposure to pesticides that oping male reproductive system in rodents. These pesti-
have a common mechanism of toxicity and to consider cides include the herbicide linuron and the fungicides
aggregate exposure, i.e., exposure from all conceivable vinclozolin, procymidone, and prochloraz. A group of
pathways (food, drinking water, residential activities) and Danish researchers provided evidence that the combination
routes (ingestion, inhalation, and dermal absorption). The of vinclozolin and procymidone [203] or a mixture of those
USEPA is specifically directed to take into account the special two pesticides plus flutamide (an antiandrogenic pharma-
susceptibility of infants and children and the behavioral ceutical) induces male reproductive malformations and
patterns that may put them at increased risk from dispropor- changes in prostate gene expression in a dose-additive
tionately high levels of exposure. manner [204–206]. According to comments made in 2000,
Following the mandate of the FQPA, the USEPA con- the USEPA has been well aware of the possibility that
ducted cumulative and aggregate risk assessments for OP vinclozolin and a subgroup of the dicarboximide class of
pesticides, N-methyl carbamate pesticides, and a subset of fungicides (which includes procymidone) may modulate
triazine pesticides that share the ability to cause neuroendo- androgens by a common mechanism of toxicity. At the
crine and endocrine-related developmental, reproductive, and time, however, it considered the available evidence insuf-
carcinogenic effects. It employed the TEF approach for the ficient to determine whether this was indeed the case.
cumulative exposure assessment, using chlorpyrifos as the Not only pesticide residues but also phthalates belong to
index compound for OP pesticides because the most the chemical compounds to which consumers are exposed
extensive data were available for this particular substance. on a daily basis. In addition, all phthalates that affect male
There are indications that the choice of the index reproductive development have been shown to do so via a
compound can have marked effects on the resulting common mechanism of action, namely a reduction in
exposure estimates [198,199]. For example, estimates of testicular testosterone production and in Leydig cell Insl3
the high end of cumulative exposure to OP and carbamate expression. When DBP and DEHP were combined, each at
pesticides in the Dutch population amounted to 13.4µg/kg half of the effective dose (ED) predicted to cause a 50%
bw per day with acephate as the index compound but incidence of epididymal agenesis, dose addition was found
27.6µg/kg bw per day with phosmet as the index to provide the best model for predicting several of the
compound [198]. If determined under ideal conditions, examined outcomes [207]. In addition to the incidence of
epididymal agenesis, these included decreases in the It is worth underscoring again that the mechanisms by
anogenital distance and the incidence of areolae retention which the various compounds induce male reproductive
and testis malformation. Note, however, that the frequen- toxicity are different. Vinclozolin and procymidone are AR
cies of hypospadias, gubernacular agenesis, and seminal antagonists, while the phthalates are not. Conversely, the
vesicle malformations were greater than predicted by dose phthalates act by reducing fetal testosterone and Insl3 mRNA
additivity, suggesting a synergistic effect of the two expression and protein production, which the two fungicides
phthalates. In an extension of this study, the male do not. Linuron affects male reproductive development by
reproductive toxicity of a mixture of five phthalates (BBP, multiple mechanisms, including AR antagonism and reduc-
DBP, DEHP, DPP, and di-isobutyl phthalate) combined at tion in fetal testosterone production, but it does not alter Insl3
their respective ED50 values was investigated [208]. Again, expression. Similarly, dose addition was found to predict the
the dose addition model accurately predicted fetal mortality effects of a mixture of PCDD/Fs, dioxin-like PCBs, and non-
and fetal testosterone production. dioxin like PCBs, which individually all disrupt thyroid
The same group of scientists, which incidentally hormone homeostasis but via different mechanisms [211].
includes researchers from the USEPA, then began investi- The fact that dose additivity can be observed with mixtures
gating the effects of phthalates combined with pesticides of compounds that act by distinct molecular mechanisms
that act as AR antagonists. Initially, they showed that but produce similar effects in a given target organ high-
exposure during the critical window from gestational days lights that regulatory agencies need to define the term
14 to 18 to the herbicide linuron and the phthalate BBP “common mechanism of action” quite broadly rather than
individually and in combination reduced testicular testos- choosing a narrow focus on identical molecular mecha-
terone production and concentrations in GD18 rat fetuses nisms when it comes to identifying compounds that need to
[209]. The greatest effect was seen with the combination. be evaluated for the cumulative risk they pose.
Whereas the herbicide and the phthalate alone were not What constitutes a “common mechanism of toxicity” has
associated with reproductive malformations at the selected been a matter of much debate, but several scientific bodies
doses, the combination induced significant incidences of endorse a rather broad definition, where the focus should be
incomplete preputial separation, cleft prepuce, cleft phallus, on a common adverse outcome in a specific target tissue
hypospadias, and vaginal pouch. A low incidence of rather than on a common molecular target or molecular
malformations of internal reproductive tissues (adhesion pathway, possibly involving the same toxic intermediate
or agenesis of ventral prostate, agenesis of seminal vesicle ((National Research Council (NRC), http://www.nap.edu/
and epididymis, undescended testis) was seen after expo- catalog/12528.html), [197]). In particular, the NRC has
sure to BBP or linuron alone but was significantly urged the USEPA to assess the human health risk resulting
increased in the mixture group. For most of these effects, from cumulative exposure not only to all the phthalates
the two antiandrogens with mixed mechanisms of action with reproductive toxicities but also pesticides such as
were found to act in a cumulative dose-additive fashion. linuron, vinclozolin, and procymidone that act by distinct
More recently, a mixture of seven antiandrogens was tested, mechanisms but result in very similar reproductive malfor-
namely vinclozolin, procymidone, linuron, and prochloraz mations. The NRC also emphasizes that, in order for
and the phthalates BBP, DBP, and DEHP [210]. After cumulative risk assessment of phthalates to become
careful construction of dose response curves for each of possible, it will be imperative to address three key issues:
these substances, using several of the typical male (a) characterize the full spectrum of phthalate metabolites,
reproductive malformations as end points, each compound (b) determine which metabolite is most appropriate for use
was used at one seventh of its respective ED100 (i.e., they as a biomarker of human exposure, and (c) identify the
would be expected to contribute equally to the overall most important sources of human phthalate exposure.
effect). The TEF model performed at least as well as the Incidentally, such an assessment would be well within
dose addition model or even better in predicting the the mandate of the FQPA, which specifically directs the
observed reproductive malformations. This is somewhat USEPA to provide for the testing of all pesticide chemicals
surprising since the TEF approach is a specific application for their estrogenic activity “or such other endocrine effects
of dose addition. In a more recently published study, the as the Administrator may designate.” In addition, the FQPA
effects of the same antiandrogens with both similar and authorizes the USEPA to “provide for the testing of any
disparate mechanisms of toxicity were tested singly and in other substance that may have an effect that is cumulative
pairs. Regardless of the combination used (two AR to an effect of a pesticide chemical if the Administrator
antagonists, two phthalates, or AR antagonist plus phtha- determines that a substantial population may be exposed to
late), the binary mixtures containing each individual such a substance” (our emphasis).
compound at a dosage level of one half of its ED50 for Not only the USEPA but also the USFDA and other
hypospadias induced dose-additive effects. governmental departments would do well to rethink their
approach to regulating food contaminants. In an article 12. Hernandez-Becerril DU, Alonso-Rodriguez R et al (2007) Toxic
posted on the USFDA website, Healthcare without Harm and harmful marine phytoplankton and microalgae (HABs) in
Mexican Coasts. J Environ Sci Health A Tox Hazard Subst
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USA is severely “balkanized.” Phthalates were used as an 13. Panel on Contaminants in the Food Chain (2009) Marine
illustration: the USFDA is responsible for food contami- biotoxins in shellfish—saxitoxin group. Scientific Opinion of
nants (including phthalates), drug ingredients (including the Panel on Contaminants in the Food Chain. EFSA J 1019,
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Clinic Rev Allerg Immunol (2010) 39:95–141

Published online: 13 November 2009

between chemicals and investigates the effects of cumula- TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Zoonoses from cows to humans [7]. This is thought to have occurred

Pathogen Occurrence Main transmission routes Symptoms Possible complications

will be exposed to these toxins. According to the main

Can be mistaken for appendicitis,

weakness in the neck and arms, paralysis

of the respiratory muscles, and death if

released by the bacterium and are that

abdominal swelling. These symptoms

headache, muscle cramping, changed

4 h to 8 days). They can progress to

vomiting, diarrhea, constipation, or

PSP The major PSP toxins belong to the saxitoxin group,

of which at least 29 congeners are known and which are

not treated with antitoxin

0.5–2 h) and include tingling sensation of the lips, mouth,

Authority (EFSA) set an acute Rfd (ARfd) of 0.5µg/kg bw.

products, milk, and dairy

Improperly canned foods,

Both the USFDA and the EFSA have a maximum limit of

80µg/100 g (or 800µg/1 kg) of PSP toxins in saxitoxin

acute exposure of 320-µg toxins or 5.3µg/kg bw in a 60-kg

adult. Since this intake is tenfold higher than the ARfd, it

NSP Karenia brevis and several other dinoflagellates (see

also Table 2) produce hemolytic and neurotoxic substances,

the latter being designated as brevetoxins. There are a total

mouth, and face, paresthesia, loss of motor control, and

Type of Toxin Sources of toxin Primary Mechanism Symptoms

PSP Saxitoxins, Alexandrium spp., Shellfish Voltage-gated Tingling of perioral area,

individuals. Subjects with hepatitis B infection are at

increased risk of hepatocellular carcinoma from aflatoxin

variety of chemical or physical means for reducing the

aflatoxin content [20,23].

secondary metabolites that are produced mainly by Peni-

ly also by isolates of Aspergillus niger [20]. The main and

wine, and poultry meat. In the dietary exposure assessment

and Norway, the main dietary source was cereal grains in

most European countries, but coffee and wine made the

major contribution to exposure in Greece and Italy,

respectively [24,25]. Dietary intake was estimated to be

taken into account in determining exposure. In a French

study on dietary mycotoxin exposure, bread was the major

source of OTA, accounting for one third of total intake [21].

containing food types. A duplicate diet study of 123 Dutch

participants indicated a mean OTA intake of 1.2 ng/kg bw

per day [26], whereas in a UK duplicate diet study, where

dietary exposure of 0.94 ng/kg bw was calculated [27].

The absorption of OTA occurs in the upper gastrointes-

liver, muscle, and fat. It has been shown to cross the

placenta in different animal species and has been detected

in human breast milk [29,30]. There are substantial

interspecies differences in serum half-lives, ranging from

1 day in mice to 21 days in monkeys and ~35 days in

indications of enterohepatic circulation.

OTA has been shown to be nephrotoxic in almost all

animals species investigated to date. In humans, OTA

nephropathy, but a causal connection has not been proven