Scientific Opinion on acryalmide in food

321 Pages·2015·3.68 MB·English

EFSA Journal 2015;13(6):4104

1
Scientific Opinion on acrylamide in food
2, 3
EFSA Panel on Contaminants in the Food Chain (CONTAM)
European Food Safety Authority (EFSA), Parma, Italy
ABSTRACT
EFSA was asked to deliver a scientific opinion on acrylamide (AA) in food. AA has widespread uses as an
industrial chemical. It is also formed when certain foods are prepared at temperatures above 120 °C and low
moisture, especially in foods containing asparagine and reducing sugars. The CONTAM Panel evaluated
43 419 analytical results from food commodities. AA was found at the highest levels in solid coffee substitutes
and coffee, and in potato fried products. Mean and 95th percentile dietary AA exposures across surveys and age
groups were estimated at 0.4 to 1.9 µg/kg body weight (b.w.) per day and 0.6 to 3.4 µg/kg b.w. per day,
respectively. The main contributor to total dietary exposure was generally the category ‘Potato fried products
(except potato crisps and snacks)’. Preferences in home-cooking can have a substantial impact on human dietary
AA exposure. Upon oral intake, AA is absorbed from the gastrointestinal tract and distributed to all organs. AA
is extensively metabolised, mostly by conjugation with glutathione but also by epoxidation to glycidamide
(GA). Formation of GA is considered to represent the route underlying the genotoxicity and carcinogenicity of
AA. Neurotoxicity, adverse effects on male reproduction, developmental toxicity and carcinogenicity were
identified as possible critical endpoints for AA toxicity from experimental animal studies. The data from human
studies were inadequate for dose-response assessment. The CONTAM Panel selected BMDL10 values of
0.43 mg/kg b.w. per day for peripheral neuropathy in rats and of 0.17 mg/kg b.w. per day for neoplastic effects
in mice. The Panel concluded that the current levels of dietary exposure to AA are not of concern with respect to
non-neoplastic effects. However, although the epidemiological associations have not demonstrated AA to be a
human carcinogen, the margins of exposure (MOEs) indicate a concern for neoplastic effects based on animal
evidence.
© European Food Safety Authority, 2015
KEY WORDS
acrylamide, glycidamide, exposure, food, risk assessment, BMD, MOE

1
On request from the European Commission, Question No EFSA-Q-2013-00007, adopted on 30 April 2015.
2
Panel members: Diane Benford, Sandra Ceccatelli, Bruce Cottrill, Michael DiNovi, Eugenia Dogliotti, Lutz Edler, Peter
Farmer, Peter Fürst, Laurentius (Ron) Hoogenboom, Helle Katrine Knutsen, Anne-Katrine Lundebye, Manfred Metzler,
Antonio Mutti (as of 6 October 2014), Carlo Stefano Nebbia, Michael O’Keeffe, Annette Petersen (as of 6 October 2014),
Ivonne Rietjens (until 2 May 2014), Dieter Schrenk, Vittorio Silano (until 21 July 2014), Hendrik van Loveren, Christiane
Vleminckx, and Pieter Wester. Correspondence:
Acrylamide in food

SUMMARY
Following a request from the European Commission, the Panel on Contaminants in the Food Chain
(CONTAM Panel) was asked to deliver a scientific opinion on acrylamide (AA) in food. The Panel
developed the draft scientific opinion which underwent a public consultation from 1 July 2014 to
15 September 2014. The comments received and how they were taken into account when finalising
the scientific opinion were published in an EFSA Technical Report (EFSA, 2015).
AA is a low molecular weight, highly water soluble, organic compound. It is used inter alia as an
industrial chemical and in the production of polyacrylamides. Heightened concerns about exposure to
AA arose in 2002 when it was discovered that it forms when certain foods are prepared at
temperatures usually above 120 °C and low moisture. It forms, at least in part, due to a Maillard
reaction between certain amino acids, such as asparagine, and reducing sugars. However, several
other pathways and precursors have also been proposed to contribute to AA formation. AA forms in
numerous baked or fried carbohydrate-rich foods, including French fries, potato crisps, breads,
biscuits and coffee. AA is also known to be present in cigarette smoke.
The analytical determination of AA in food products is most frequently performed by high
performance liquid chromatographic (HPLC) or gas chromatographic (GC) separation methods with
mass spectrometric detection (MS), either in selected ion monitoring (SIM) mode or by tandem mass
spectrometry (MS/MS) in multiple reaction mode (MRM) using isotope labelled standards.
In its exposure assessment, the CONTAM Panel evaluated a total of 43 419 analytical results from
food commodities collected and analysed since 2010 and reported by 24 European countries and
six food associations. Data provided by European countries and those provided by food associations
gave overall consistent and complementary information. AA was found at the highest levels in
‘Coffee substitutes (dry)’ (average medium bound (MB) levels of 1 499 µg/kg) and ‘Coffee (dry)’
(average medium bound (MB) levels of 522 µg/kg). However, due to dilution effects, lower levels are
expected in ‘Coffee beverages’ and ‘Coffee substitutes beverage’ as consumed by the population.
High levels were also found in ‘Potato crisps and snacks’ (average MB level of 389 µg/kg) and
‘Potato fried products (except potato crisps and snacks)’ (average MB level of 308 µg/kg). Lower AA
levels were found in ‘Processed cereal-based baby foods’ (average MB level of 73 µg/kg), ‘Soft
bread’ (average MB level of 42 µg/kg) and ‘Baby foods, other than cereal-based’ (average MB level
of 24 µg/kg). The CONTAM Panel explored the possibility of performing a temporal trend analysis of
the AA concentrations in certain foodstuffs across Europe on the basis of the data submitted to EFSA
by the European countries. Because of gaps in the databases and the fact that results for different years
are not always comparable, a reliable Europe-wide temporal trend analysis is not feasible. However, a
dataset of manufacturers’ measurements of AA levels in 40 455 samples of fresh sliced potato crisps
from 20 European countries for the years 2002 to 2011 showed a substantial downward trend for
mean levels of AA, from 763 ± 91.1 µg/kg in 2002 to 358 ± 2.5 µg/kg in 2011. For other food
categories, a similar downward trend was not observed.
Estimation of human exposure to AA revealed that infants, toddlers and other children were the most
exposed groups. Depending on the survey and age group, chronic dietary exposure of children was
estimated to be on average between 0.5 and 1.9 µg/kg b.w. per day and the 95th percentile was
between 1.4 and 3.4 µg/kg b.w. per day. Chronic dietary exposure of adolescents, adults, elderly and
very elderly was estimated to be on average between 0.4 and 0.9 µg/kg b.w. per day and the
95th percentile was between 0.6 and 2.0 µg/kg b.w. per day depending on the survey and age group.
The main contributor to the total AA exposure of infants was ‘Baby foods, other than processed
cereal-based’ followed by ‘Other products based on potatoes’ and ‘Processed cereal-based baby
foods’. The main contributor to the total exposure of toddlers, other children and adolescents was
‘Potato fried products (except potato crisps and snacks)’ representing up to half the total exposure,
followed by ‘Soft bread’, ‘Breakfast cereals’, ‘Biscuits, crackers, crisp bread’, ‘Other products based
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on cereals’ and ‘Other products based on potatoes’. These foods groups were also the main
contributors to the total exposure of adults, elderly and very elderly together with ‘Coffee’.
Scenarios were designed in order to assess the influence of specific behaviours (preference for
particular products, places of consumption, home-cooking habits) on the total dietary exposure to AA.
Scenarios on the preference for particular potato crisps and coffee products resulted in a variation of
less than 4 % and 14 %, respectively, of the total dietary exposure to AA. In scenarios on home-
cooking behaviours, degree of bread toasting resulted in variations of less than 8 %, while for
conditions of potato frying the total dietary exposure to AA could be increased up to 80 %.
In both experimental animals and humans upon oral intake, AA that is not covalently bound to
components of the food matrix is extensively absorbed from the gastrointestinal tract. After reaching
the systemic circulation, AA is rapidly distributed into the tissues. AA is also able to cross the
placenta and is transferred to a small extent into human milk. AA is extensively metabolised, mostly
by conjugation with glutathione (GSH) (primarily mediated by glutathione-S-transferases) but also by
epoxidation to glycidamide (GA), which is widely distributed into tissues. The formation of GA
represents a metabolic activation pathway preferentially mediated by CYP2E1. Mice are more
proficient in converting AA into GA than either rats or humans.
Formation of GA is considered to represent the route underlying the genotoxicity and carcinogenicity
of AA. Covalent DNA adducts of GA have been amply demonstrated in in vitro models and
experimental animals, and these have been used as biomarkers of AA exposure. The N7-guanine
adduct derived from GA (N7-GA-Gua) is the most abundant DNA adduct following AA exposure.
GA-DNA adducts in experimental animals are found at similar levels in various tissues of the body,
although CYP2E1 is primarily located in the liver.
Covalent adducts of AA with DNA have been generated in chemical reactions, but have never been
detected in vivo or in vitro in animal or human tissues.
Detoxification of both AA and GA can proceed through conjugation with GSH and the GSH adducts
are subsequently converted to mercapturic acids, which are excreted in urine. The mercapturic acids
of AA and GA represent the major metabolites and their urinary excretion levels can be used as
biomarkers of AA exposure. AA and GA can also react with proteins to form covalent adducts, e.g.
with haemoglobin (Hb). Also these Hb adducts represent important biomarkers of AA exposure.
Several studies have reported various approaches to physiologically based pharmacokinetic (PBPK)
modelling of AA absorption, metabolism, and disposition with the goal of predicting human internal
exposures to AA and GA (i.e. area under the curve, AUC) for use in reducing the uncertainty in risk
assessment inherent in animal to human extrapolations.
PBPK models allow derivation of human-equivalent doses (HEDs), which could be used to convert
external doses of AA that produce critical effects in animal studies to the human external doses
required to produce equivalent AUC values for either AA or GA, depending on the toxic endpoint
used. The HEDs derived from equivalent AA-AUCs in rats and mice suggest that endpoints related to
AA-mediated effects (e.g. neurotoxicity) require 4- to 6-fold higher doses in rats when compared to
humans, based on inter-species differences in toxicokinetics. However, 0.5- to 0.7-fold lower doses of
AA would be required in mice to produce equivalent GA-AUCs for genotoxicity-related endpoints
when compared to humans.
Toxicological studies with AA have been conducted in rats, mice, monkeys, cats and dogs, using
various dosing protocols and routes of exposure. Oral LD50 values for AA were reported to be
> 150 mg/kg b.w. for rats, 107 mg/kg b.w. for mice, and 150–180 mg/kg b.w. for rabbits and guinea
pigs.
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Adverse effects reported in repeated dose toxicity studies of AA in rats, mice, monkeys, cats and dogs
consisted of loss of body weight and effects on the nervous system reflected by hind-limb paralysis,
reduction in rotorod performance and/or histopathological changes in peripheral nerves and nervous
system structures. In mice, effects reported in addition to the neurotoxicity consisted of effects on the
testes, including the degeneration of epithelia in spermatids and spermatocytes, the reduction of
spermatozoa, and the presence of multinucleate giant cells, as well as forestomach hyperplasia,
hematopoietic cell proliferation of the spleen and preputial gland inflammation, lung alveolar
epithelium hyperplasia and cataract and for female mice ovarian cysts.
In rats, effects reported in addition to the neurotoxicity, included atrophy of skeletal muscle, testicular
atrophy, distended urinary bladders, increased prevalence of duct ectasia in preputial glands,
hematopoietic cell proliferation in the spleen, bone marrow hyperplasia, ovarian atrophy, degeneration
of the retina, exfoliated germ cells epididymis, hepatocyte degeneration and liver necrosis, bone
marrow hyperplasia, mesenteric lymph node cellular infiltration and pituitary gland hyperplasia.
Thirteen-week and 2-year studies in mice and rats dosed with GA revealed adverse effects that were
generally similar to those reported for AA. Rats were more sensitive to the neurotoxic effects of AA
and GA than mice, and neurotoxicity in rats (such as hind-leg paralysis and peripheral neuropathy)
was consistently associated with lower AA doses and greater severity when compared to equimolar
concentrations of GA.
Rodent studies have demonstrated adverse effects of AA on male reproductive parameters including
reduced sperm counts and effects on sperm and testis morphology with a no-observed-adverse-effect
level (NOAEL) of approximately 2 mg/kg b.w. per day.
Rat and mouse studies have shown some signs of developmental toxicity (increased incidence of
skeletal variations, slightly impaired body weight gain, histological changes in the central nervous
system, and neurobehavioural effects) at exposure levels that in some cases are also associated with
maternal toxicity. The lowest NOAEL reported for developmental toxicity was 1.0 mg/kg b.w. per
day from studies in rats exposed gestationally and neonatally.
The genotoxicity of AA, as well as of its reactive metabolite GA, has been extensively studied. In
vitro genotoxicity studies indicate that AA is a weak mutagen in mammalian cells but an effective
clastogen. GA is a strong mutagen and a clastogen. It induces mutations via a DNA adduct
mechanism. In vivo, AA is clearly genotoxic in somatic and germ cells. AA exerts its mutagenicity via
metabolism by CYP2E1 to GA. AA can also induce gene mutations by a pathway involving the
generation of reactive oxygen species (ROS) and oxidative DNA damage.
AA is carcinogenic in multiple tissues in both male and female mice and rats. In rats, the major
tumours produced by AA are adenomas, fibroadenomas and fibromas of the mammary gland, thyroid
gland follicular cell adenomas or carcinomas, and in F344 rats, testes or epididymis tunica vaginalis
mesotheliomas. In mice, the major tumours produced by AA are: Harderian gland adenomas,
mammary gland adenoacanthomas and adenocarcinomas, lung alveolar and bronchiolar adenomas,
benign ovary granulosa cell tumours, skin sarcomas, and stomach and forestomach squamous cell
papillomas in females, and Harderian gland adenomas and adenocarcinomas, lung alveolar and
bronchiolar adenomas and carcinomas, and stomach squamous papillomas and carcinomas in males.
A similar spectrum of tumours is observed when equimolar concentrations of GA were administered
in drinking water to rats and mice, which is consistent with GA being the proximate carcinogenic
metabolite of AA.
AA is an electrophilic molecule which can undergo Michael addition-type reactions with nucleophilic
target molecules. In particular, activated thiolate moieties in cysteine residues of enzymes and other
functional proteins, e.g. in neuronal cells or spermatocytes, have been described as targets. The
neurotoxic properties of AA are considered to originate mainly from this type of reactivity.
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AA shows some reactivity towards nucleic acids, whereas reports on the formation of DNA adducts in
vivo suggest that GA is mainly, if not exclusively, responsible for the formation of DNA adducts in
AA-treated animals.
Evidence from the available studies in the literature on hormonal and endocrine effects of AA is
equivocal. This is particularly true for changes in hormone levels in AA-treated animals which were
reported in some studies. Mechanistic hypotheses on local endocrine effects of AA which may explain
tumour formation in certain hormone or paracrine-regulated target tissues lack experimental proof.
There is a wide range of epidemiological studies investigating possible effects of AA in humans. Two
cohort studies considered occupational exposure to AA and did not indicate an increased cancer risk.
Associations between AA exposure through diet and cancer risk have been analysed in at least
36 publications, based on 16 epidemiological studies considering several cancer sites.
For most cancers there is no consistent indication for an association between AA exposure and
increased risk. A few studies suggested an increased risk for renal cell, and endometrial (in particular
in never-smokers) and ovarian cancer, but the evidence is limited and inconsistent. Moreover, one
study suggested a lower survival in non-smoking women with breast cancer with a high pre-diagnostic
exposure to AA but more studies are necessary to confirm this result.
Two studies reported an inverse relation between AA exposure (measured by levels of AA and GA
adducts) and birth weight and other markers of fetal growth. The CONTAM Panel noted that it has
not been established whether the association between dietary AA exposure and these outcomes is
causal.
Studies among workers occupationally exposed to AA showed an increased risk of neurological
alterations, including mostly the peripheral, but also the central nervous system.
From all data available, the CONTAM Panel identified four possible critical endpoints for AA
toxicity, i.e. neurotoxicity, effects on male reproduction, developmental toxicity, and carcinogenicity.
The data from human studies were not adequate for dose-response assessment. Therefore, the
CONTAM Panel considered the data from studies on experimental animals to establish the reference
points. The CONTAM Panel performed benchmark dose (BMD) analyses on data for neurotoxicity
and on the tumour incidences induced by AA in experimental animals. The CONTAM Panel selected
the value of 0.43 mg/kg b.w. per day derived as the lowest BMDL10 from the data on incidences of
peripheral nerve (sciatic) axonal degeneration in male F344 rats exposed to AA in drinking water for
two years as the reference point for non-neoplastic effects. Based on the fact that this BMDL10 is
lower than the NOAEL of approximately 2 mg/kg b.w. per day for adverse effects on male
reproductive parameters and of 1.0 mg/kg b.w. per day for developmental toxicity, the CONTAM
Panel concluded that using the BMDL10 for neurotoxicity as the reference point is conservative when
considering possible non-neoplastic effects of AA.
For neoplastic effects, the CONTAM Panel selected as a reference point the value of 0.17 mg/kg b.w.
per day derived as the lowest BMDL10 from data on incidences of Harderian gland adenomas and
adenocarcinomas in male B6C3F1 mice exposed to AA for two years. The CONTAM Panel noted that
the Harderian gland is an organ absent in humans, but that in rodents this organ is a sensitive target
tissue to detect compounds that are both genotoxic and carcinogenic. Taking into account that target
tissues for tumour formation by a given genotoxic carcinogen may differ between rodents and
humans, the CONTAM Panel considered the most sensitive target tissue in rodent bioassays, the
Harderian gland, a conservative endpoint for assessment of the risk for neoplastic effects of AA in
humans.
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The fact that AA and its metabolite GA are positive in a variety of genotoxicity tests indicates that AA
is of concern with respect to genotoxicity. Therefore, the CONTAM Panel considered it inappropriate
to establish a tolerable daily intake (TDI).
Risk characterisation for non-neoplastic effects was performed using the margin of exposure (MOE)
approach and the BMDL10 value of 0.43 mg/kg b.w. per day for the most relevant and sensitive
endpoint for neurotoxicity, i.e. the incidence of peripheral nerve (sciatic) axonal degeneration
observed in F344 rats exposed to AA in drinking water for two years in the NTP study. MOE values
for the neurotoxic effects ranged from 1 075 (minimum LB) to 226 (maximum UB) for the mean
exposure, and from 717 (minimum LB) to 126 (maximum UB) for the 95th percentile exposure
estimates across surveys and age groups). Taking into account differences between species and within
the human population, the Panel concluded that the MOEs across surveys and age groups are not of
concern. However, the Panel noted that the MOEs for the 95th percentile UB exposure estimates for
toddlers and other children are close to the value that might be of concern for neurotoxicity.
For the risk characterisation for neoplastic effects, the MOE approach for compounds that are both
genotoxic and carcinogenic is considered appropriate, using as the reference point the BMDL10 of
0.17 mg/kg b.w. per day, i.e. the lowest BMDL10 from data on incidences of Harderian gland
adenomas and adenocarcinomas in male B6C3F1 mice exposed to AA for two years in the NTP study.
Comparison of the data on human exposure levels to AA across surveys and age groups reported
above to this BMDL10 of 0.17 mg/kg b.w. per day, reveals MOE values that range from
425 (minimum LB) to 89 (maximum UB) for the mean exposure estimates, and from 283 (minimum
LB) to 50 (maximum UB) for the 95th percentile exposure estimates across all surveys and age
groups. The EFSA Scientific Committee concluded that, for substances that are both genotoxic and
carcinogenic, an MOE of 10 000 or higher, based on a BMDL10 from an animal study, and taking into
account overall uncertainties in the interpretation, would be of low concern from a public health point
of view. Since the MOEs calculated are all substantially lower than the value of 10 000, the
CONTAM Panel concluded that, although the available human studies have not demonstrated AA to
be a human carcinogen, the MOEs across surveys and age groups indicate a concern with respect to
neoplastic effects.
The CONTAM Panel noted that AA is a germ cell mutagen and that there are at present no established
procedures for risk assessment using this endpoint.
Finally, the CONTAM Panel makes the following recommendations: the reporting of AA occurrence
data should be improved regarding the mode of preparation of the products before analysis. Duplicate
diet studies are recommended in order to improve exposure assessment, since they provide a more
accurate indication of AA levels in food as prepared and consumed at home. Data on urinary
metabolites levels from individuals participating in the duplicate diet studies should be generated for
the purpose of validation of the biomarkers. An up-to-date OECD compliant extended one-generation
or two-generation reproductive toxicity study investigating the effects of AA on sperm parameters and
including a detailed histopathological examination of the testis and accessory glands as well as
investigating the effects on development until puberty should be conducted. If further epidemiological
studies are conducted to assess possible associations between dietary AA intake and risk of cancers
(e.g. endometrium, ovary and renal cells), they should have improved measurement of AA exposure,
and should be sufficiently powered. Further epidemiological studies are required to confirm or refute
the inverse relation between dietary AA intake and birth weight and other markers of fetal growth
observed in two studies. Improved approaches for the detection and risk assessment of germ cell
mutagens should be developed, and applied to AA and GA.
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TABLE OF CONTENTS
Abstract .................................................................................................................................................... 1
Summary .................................................................................................................................................. 2
Background as provided by the European Commission ......................................................................... 10
Terms of reference as provided by the European Commission .............................................................. 10
Assessment ............................................................................................................................................. 11
1. Introduction ................................................................................................................................... 11
1.1. Previous risk assessments ..................................................................................................... 12
1.2. Chemical characteristics ....................................................................................................... 23
1.3. Production, use and environmental fate ................................................................................ 23
1.3.1. Industrial production and use............................................................................................ 23
1.3.2. Environmental fate ........................................................................................................... 24
1.3.3. Formation in food ............................................................................................................. 24
2. Legislation ..................................................................................................................................... 25
3. Sampling and methods of analysis ................................................................................................ 28
3.1. Sample collection and frequency .......................................................................................... 28
3.2. Methods of analysis .............................................................................................................. 28
3.2.1. Legal requirements ........................................................................................................... 28
3.2.2. Analytical approaches ....................................................................................................... 29
3.2.2.1. Food ......................................................................................................................... 29
3.2.2.2. Biological matrices .................................................................................................. 30
3.2.3. Analytical quality assurance: reference materials, validation and proficiency testing ..... 31
4. Occurrence and patterns of AA in food ......................................................................................... 32
4.1. Current occurrence of AA in food – Occurrence results reported to EFSA ......................... 32
4.1.1. Overview of the datasets ................................................................................................... 32
4.1.2. Data management and validation...................................................................................... 32
4.1.2.1. Sampling requirements ............................................................................................ 32
4.1.2.2. Analytical requirements ........................................................................................... 32
4.1.2.3. Food description ...................................................................................................... 33
4.1.2.4. Data submitted by European countries .................................................................... 33
4.1.2.5. Data submitted by food associations ........................................................................ 34
4.1.2.6. Comparison of both datasets .................................................................................... 35
4.1.3. Description of the occurrence levels ................................................................................. 39
4.1.3.1. Potato fried products (except potato crisps and snacks) .......................................... 41
4.1.3.2. Potato crisps and snacks........................................................................................... 42
4.1.3.3. Soft and crisp bread ................................................................................................. 42
4.1.3.4. Breakfast cereals ...................................................................................................... 42
4.1.3.5. Biscuits, cracker and gingerbread ............................................................................ 42
4.1.3.6. Coffee (dry) .............................................................................................................. 42
4.1.3.7. Coffee substitutes (dry) ............................................................................................ 43
4.1.3.8. Baby foods ............................................................................................................... 43
4.2. Temporal trend analysis of AA occurrence data in certain food categories ......................... 43
4.3. Previously reported literature data on AA in food ................................................................ 44
4.3.1. Glycidamide in food ......................................................................................................... 46
4.3.2. Data published by consumer organisations and consumer’s magazines .......................... 46
4.4. Impact of raw material, storage and processing on AA levels in food ................................. 47
4.4.1. Impact of raw material and storage .................................................................................. 47
4.4.2. Impact of processing ......................................................................................................... 48
4.4.3. Concluding remarks .......................................................................................................... 52
4.5. Initiatives for mitigation measures ........................................................................................ 52
5. Food consumption ......................................................................................................................... 54
5.1. EFSA’s Comprehensive European Food Consumption Database ........................................ 54
5.2. Specific consumption patterns of the total population and of ‘consumers-only’ in European
countries ............................................................................................................................................. 57
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5.2.1. French fries and potato fried ............................................................................................. 57
5.2.2. Potato crisps ...................................................................................................................... 57
5.2.3. Coffee ............................................................................................................................... 57
6. Human exposure assessment ......................................................................................................... 58
6.1. Methodology ......................................................................................................................... 58
6.1.1. Food grouping................................................................................................................... 58
6.1.2. Exposure calculation ......................................................................................................... 59
6.1.3. Exposure scenarios ........................................................................................................... 60
6.1.3.1. Baseline exposure scenario ...................................................................................... 60
6.1.3.2. Scenarios reflecting specific consumption habits .................................................... 60
6.2. Results ................................................................................................................................... 60
6.2.1. Acrylamide exposure levels across the different population groups ................................ 60
6.2.1.1. General population ................................................................................................... 60
6.2.1.2. Specific population groups ...................................................................................... 61
6.2.2. Food groups contributing to the total AA exposure of the general population ................ 61
6.2.3. Exposure levels resulting from home cooking, preference for particular products and
places of consumption ................................................................................................................... 65
6.2.3.1. Home-cooking habits and places of consumption ................................................... 65
6.2.3.2. Preference for particular products ............................................................................ 68
6.2.4. Considerations on unspecified and/or raw potato products .............................................. 69
6.3. Previously reported human exposure assessments ................................................................ 69
6.3.1. General population (adults) .............................................................................................. 70
6.3.2. Infants, toddlers, other children and adolescents .............................................................. 71
6.3.3. Comparison between dietary exposure estimates made in this opinion and previously
reported exposure assessments ...................................................................................................... 72
6.4. Potential non-dietary sources of exposure ............................................................................ 73
7. Hazard identifications and characterisation ................................................................................... 74
7.1. Toxicokinetics ....................................................................................................................... 74
7.1.1. Absorption and distribution .............................................................................................. 74
7.1.2. Metabolism ....................................................................................................................... 79
7.1.2.1. Metabolic pathways ................................................................................................. 79
7.1.2.2. Enzymology ............................................................................................................. 81
7.1.3. Excretion ........................................................................................................................... 85
7.1.4. Conclusion on absorption, distribution, metabolism and excretion of AA....................... 87
7.1.5. Physiologically Based PharmacoKinetic (PBPK) modelling ........................................... 88
7.1.5.1. Comparisons of PBPK model simulations of internal dosimetry ............................ 90
7.1.5.2. Inter-species extrapolation of dosimetrics for AA and GA using a Human-
Equivalent Dose (HED) approach. ............................................................................................ 91
7.1.5.3. Use of PBPK modelling for human cancer and neuropathy risk assessments ......... 92
7.2. Biomarkers of exposure/effects ............................................................................................ 93
7.2.1. Mercapturic acids ............................................................................................................. 94
7.2.1.1. Analytical methods .................................................................................................. 94
7.2.1.2. Use of mercapturic acids as biomarkers .................................................................. 94
7.2.2. Hb adducts ........................................................................................................................ 96
7.2.2.1. Analytical methods .................................................................................................. 96
7.2.2.2. Use of Hb adducts as biomarkers ............................................................................. 97
7.2.3. DNA adducts .................................................................................................................. 101
7.2.3.1. Analytical methods ................................................................................................ 102
7.2.3.2. Use of DNA adducts as biomarkers ....................................................................... 102
7.2.4. Correlation between biomarkers ..................................................................................... 103
7.2.5. Conclusion ...................................................................................................................... 104
7.3. Toxicity in experimental animals ........................................................................................ 105
7.3.1. Acute toxicity ................................................................................................................. 105
7.3.2. Repeated dose toxicity .................................................................................................... 105
7.3.2.1. Mice ....................................................................................................................... 105
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7.3.2.2. Rats ........................................................................................................................ 107
7.3.2.3. Cats ........................................................................................................................ 112
7.3.2.4. Dogs ....................................................................................................................... 113
7.3.2.5. Hamsters ................................................................................................................ 113
7.3.2.6. Monkeys ................................................................................................................. 113
7.3.2.7. Summary of repeated dose toxicity ........................................................................ 114
7.3.3. Genotoxicity ................................................................................................................... 115
7.3.3.1. In vitro genotoxicity of AA and GA ...................................................................... 115
7.3.3.2. In vivo genotoxicity of AA and GA ....................................................................... 127
7.3.3.3. Conclusions ............................................................................................................ 140
7.3.4. Long-term toxicity and carcinogenicity .......................................................................... 141
7.3.4.1. Studies in mice ....................................................................................................... 141
7.3.4.2. Studies in rats ......................................................................................................... 143
7.3.4.3. Conclusions ............................................................................................................ 145
7.3.5. Reproductive and developmental toxicity ...................................................................... 146
7.3.5.1. Reproductive toxicity ............................................................................................. 146
7.3.5.2. Developmental toxicity .......................................................................................... 152
7.3.6. Mechanisms and modes of action ................................................................................... 158
7.3.6.1. Chemical reactivity of AA ..................................................................................... 158
7.3.6.2. Mode of action of neurotoxicity ............................................................................ 158
7.3.6.3. Mode of action of genotoxicity .............................................................................. 159
7.3.6.4. Modes of action of carcinogenicity ........................................................................ 161
7.3.6.5. Mode of action of thyroid toxicity ........................................................................ 163
7.3.6.6. Mode of action of reproductive toxicity ................................................................ 164
7.3.6.7. Mode of action of accompanying effects ............................................................... 166
7.3.6.8. Conclusions ............................................................................................................ 167
7.4. Observations in humans ...................................................................................................... 167
7.4.1. Epidemiological studies: cancer ..................................................................................... 167
7.4.1.1. Occupational studies and cancer ............................................................................ 167
7.4.1.2. Dietary studies and cancer ..................................................................................... 168
7.4.2. Epidemiological studies: pre-natal exposure .................................................................. 185
7.4.2.1. Developmental consequences ................................................................................ 185
7.4.2.2. Effects on gene expression ..................................................................................... 186
7.4.3. Epidemiological studies: neurological alterations .......................................................... 187
7.5. Considerations of critical effects and possibilities for derivation of a health based guidance
value ............................................................................................................................................ 190
7.5.1. Considerations of critical effects .................................................................................... 190
7.5.2. Dose-response assessment .............................................................................................. 190
7.5.3. Possibilities for derivation of a health based guidance value ......................................... 192
8. Risk characterisation .................................................................................................................... 192
9. Uncertainty .................................................................................................................................. 194
9.1. Assessment objectives ........................................................................................................ 195
9.2. Exposure scenarios/Exposure models ................................................................................. 195
9.3. Model input (parameters) .................................................................................................... 196
9.4. Other uncertainties .............................................................................................................. 196
9.5. Summary of uncertainties ................................................................................................... 196
Conclusions and recommendations ...................................................................................................... 197
Documentation provided to EFSA ....................................................................................................... 204
References ............................................................................................................................................ 204
Appendices ........................................................................................................................................... 247
Abbreviations ....................................................................................................................................... 317
EFSA Journal 2015;13(6):4104 9
Acrylamide in food

BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION
Acrylamide is a carcinogenic substance that is formed in foods that have undergone heat treatment,
e.g. roasting, baking and frying, and that are high in certain amino acids and reducing sugars.
Acrylamide is therefore an issue in French fries and potato crisps, but also in other foodstuff such as
biscuits, coffee, etc.
4
Industry (Food and Drink Europe) have developed a so-called ‘toolbox’ containing measures that can
be applied by the different sectors of food industry to bring acrylamide levels down. Sector specific
brochures have also been developed.
5
The Commission is monitoring acrylamide levels in food via specific monitoring recommendations.
The results of the monitoring are compiled by EFSA. Despite the fact that the industry toolbox i s in
place since 2006, levels in food are not systematically decreasing in all concerned food commodities,
as demonstrated by the results compiled since 2007.
6
Therefore a second Commission recommendation was adopted in January 20 11 which asks Member
State s to carry out further investigations at food operator’s premises in case high acrylamide levels are
found. Indicative values have been established in that recommendation. If an indicative value is
exceeded, an investigation should be carried out. The indicative values are not legal limits and do not
require enforcement action if they are exceeded.
In the near future, the Commission will assess the approach taken and decide about the need for
further appropriate measures.
However, since the Statement of the Scientific Panel on Contaminants in the Food Chain to a
summary report on acrylamide in food of the 64th meeting of the Join FAO/WHO Expert Committee
on Food Additives (Adopted on 19 April 2005) and the ESFA Scientific Colloquium on acrylamide
7
carcinogenicity, new scientific information had become available.
In order to assess the need for further measures as regards acrylamide in food, EFSA is requested to
assess the risk related to the presence of acrylamide in food.
TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION
In accordance with Art 29 (1) of Regulation (EC) No 178/2002, the European Commission asks the
European Food Safety Authority for a scientific opinion on the risk to human health related to the
presence of acrylamide in food.

4
Food and Drink Europe Acrylamide toolbox available at: http://ec.europa.eu/food/food/chemicalsafety/contaminants/
ciaa_acrylamide_toolbox09.pdf
5
Commission Recommendation 2007/33/EC on the monitoring of acrylamide lev els in food (OJ L123, 12.5.2007, p. 33) and
Commission Recommendation 2010/307/EU on the monitoring of acrylamide levels in food (OJ L137, 3.6.2010, p. 4)
6
Commission Recommendation of 10.1.2011 on investigations into the levels of acrylamide in food (do cument C(2010)
9681 final, available at: http://ec.europa.eu/food/food/chemicalsafety/contaminants/recommendation_10012011_
acrylamide_food_en.pdf
7
EFSA Scientific Colloquium no. 11 on acrylamide carcinogenicity – new evidence in relation to dietary exposure, held at
Tabiano (PR), Italy from 22–23 May 2008.
EFSA Journal 2015;13(6):4104 10