Alternaria Toxins in Feed and Food

EFSA Journal 2011;9(10):2407
SCIENTIFIC OPINION
Scientific Opinion on the risks for animal and public health related to the
presence of Alternaria toxins in feed and food1
EFSA Panel on Contaminants in the Food Chain (CONTAM)2, 3
European Food Safety Authority (EFSA), Parma, Italy
ABSTRACT
The European Commission asked the European Food Safety Authority to review the safety of Alternaria toxins
in food and feed. In addition to causing plant diseases on many crops such as cereals, oilseeds, tomatoes, apples
and olives, some of these toxins are genotoxic in vitro and/or fetotoxic in rats. This opinion deals with
alternariol, alternariol monomethyl ether, tenuazonic acid, iso-tenuazonic acid, altertoxins, tentoxin, altenuene
and AAL-toxins (Alternaria alternata f. sp. lycopersici toxins). Two Member States provided 11,730
occurrence results in food and also data published in the scientific literature were considered. Generally these
toxins were found in grains and grain-based products, tomato and tomato products, sunflower seeds and
sunflower oil, fruits and fruit products, and beer and wine. The feed occurrence data (1150 results) were
collected from the literature only. The knowledge on toxic effects of Alternaria toxins on farm and companion
animals and occurrence data in feed were insufficient to assess the health risk for different species. For chicken
there are indications that alternariol represents a health risk but it cannot be excluded that tenuazonic acid could
also be of concern. Considering: (1) there are few or no relevant toxicity data on Alternaria toxins, (2) the
chemical structure of several of them is known, (3) dietary exposure data exist for some of them, the Panel on
Contaminants in the food chain used the threshold of toxicological concern (TTC) approach to assess the
relative level of concern for dietary exposure of humans to these mycotoxins. For the genotoxic Alternaria
toxins, alternariol and alternariol monomethyl ether, the estimated chronic dietary exposure exceeded the
relevant TTC value indicating a need for additional toxicity data. The dietary exposure estimates for nongenotoxic tentoxin and tenuazonic acid are lower than the relevant TTC value and are considered unlikely to be
a human health concern.
European Food Safety Authority, 2011
KEY WORDS
Mycotoxins, Alternaria toxins, food, feed, analysis, occurrence, dietary exposure, animal exposure, risk
assessment, toxicity
1
On request from the European Commission, Question No EFSA-Q-2010-00960, adopted on 6 October 2011.
Panel members: Jan Alexander, Diane Benford, Alan Boobis, Sandra Ceccatelli, Bruce Cottrill, Jean-Pierre Cravedi,
Alessandro Di Domenico, Daniel Doerge, Eugenia Dogliotti, Lutz Edler, Peter Farmer, Metka Filipi, Johanna FinkGremmels, Peter Frst, Thierry Gurin, Helle Katrine Knutsen, Miroslav Machala, Antonio Mutti, Josef Schlatter, Martin
Rose and Rolaf van Leeuwen. Correspondence: contam@efsa.europa.eu
3
Acknowledgement: The Panel wishes to thank the members of the Working Group on Alternaria toxins: Jean-Pierre
Cravedi, Eugenia Dogliotti, Peter Farmer, Thierry Gurin, Manfred Metzler, Elke Rauscher-Gabernig, Carlos van
Peteghem and Antonio Vicent Civera for the preparatory work on this scientific opinion, and EFSA staff Gina Cioacata,
Valeriu Curtui, Mari Eskola and Claudia Heppner for the support provided to this scientific opinion. The CONTAM Panel
acknowledges all European competent authorities and other stakeholders that provided occurrence data on Alternaria
toxins for food and feed, and supported the consumption data collection for the Comprehensive European Food
Consumption Database.
Suggested citation: EFSA on Contaminants in the Food Chain (CONTAM); Scientific Opinion on the risks for animal and
public health related to the presence of Alternaria toxins in feed and food. EFSA Journal 2011;9(10):2407. [97 pp.]
doi:10.2903/j.efsa.2011.2407. Available online: www.efsa.europa.eu/efsajournal
2
European Food Safety Authority, 2011
Alternaria toxins in feed and food
SUMMARY
Alternaria toxins are mycotoxins produced by Alternaria species that cause plant diseases on many
crops. They are the principal contaminating fungi in wheat, sorghum and barley, and have also been
reported to occur in oilseeds such as sunflower and rapeseed, tomato, apples, citrus fruits, olives and
several other fruits and vegetables. Alternaria alternata is the most common Alternaria species in
harvested fruits and vegetables, and is the most important mycotoxin-producing species. Due to their
growth even at low temperature, Alternaria species are also responsible for spoilage of these
commodities during refrigerated transport and storage.
Alternaria species produce more than 70 phytotoxins, but a small proportion of them have been
chemically characterised and reported to act as mycotoxins to humans and animals. Some toxins such
as alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA) and altertoxins
(ATX) are described to induce harmful effects in animals, including fetotoxic and teratogenic effects.
Culture extracts of A. alternata as well as individual mycotoxins such as AOH and AME are
mutagenic and clastogenic in various in vitro systems. In addition, it has been suggested that in certain
areas in China Alternaria toxins in grains might be responsible for oesophageal cancer. Hence, due to
their possible harmful effects, Alternaria toxins are of concern for public health. They have not been
reported to cause animal toxicosis as a result of exposure from feed.
The European Commission (EC), in order to enable it to consider the need for possible follow up
actions, including filling of the knowledge gaps, asked the European Food Safety Authority (EFSA)
to provide a scientific opinion on the risks for public health related to the presence of Alternaria
toxins in feed and food.
There are no previous risk assessments on Alternaria toxins in food and feed carried out at the
European or international level. Currently there are no regulations on Alternaria toxins in food and
feed in Europe or in other regions of the world. Since AOH, AME, TeA, iso-TaA, ATXs, tentoxin
(TEN), altenuene (ALT) and Alternaria alternata f. sp. lycopersici toxins (AAL-toxins) have been
chemically characterised and they have been reported to occur in food and feed this Scientific Opinion
only considers these Alternaria toxins. However, several other Alternaria toxins have been identified.
Several chromatography based techniques are suitable for Alternaria toxin quantification in foods and
feeds, and liquid chromatography coupled to (tandem) mass spectrometry has become the method of
choice. However, there are several limiting factors for the analysis of Alternaria toxins such as the
efficiency of sample cleanup, the availability of (sufficient) amounts of standards and the lack of
reference materials for food and feed. Most of the analytical methods are to a certain extent in-house
validated but interlaboratory validation studies, standardisation of the analytical methods or
conduction of proficiency tests have not been reported.
A total of 11730 results on AOH (n = 2291), AME (n = 2215), ALT (n = 1747), ATX-I (n = 1279),
TeA (n = 1947), TEN (n = 1388) and sum of AAL-toxins (n = 863) in food were used in the
assessment. These include the data reported by two Member States (84 %) and literature data reported
for Europe (16 %). The reported data on Alternaria toxins in food were characterised by a high
proportion of left-censored data (results below the limit of detection (LOD)/limit of quantification
(LOQ)) ranging from 87 up to 100 % for the different compounds.
In samples containing Alternaria toxins, AOH, AME, TeA and TEN were generally found in certain
grains and grain-based products, tomato and tomato products, sunflower seeds and sunflower oil,
fruits and fruit products including fruit juices, and in beer and wine.
EFSA Journal 2011;9(10):2407
For feed, since no data on Alternaria toxins were submitted to EFSA, the occurrence data for feed and
agricultural commodities were collected from the available literature only. Based on studies on
occurrence of Alternaria toxins in different regions of the world, a total of 1150 results on AOH
(n = 755), AME (n = 158), ALT (n = 129) and TeA (n = 108) in feed were used in the evaluation. The
results on feed were characterised by a high proportion of left-censored data from 9 % up to 66 % for
different Alternaria toxins.
The EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) used a lower bound-upper
bound (LB-UB) approach in its assessment of the occurrence data for food and feed. The lower bound
assigns a value of zero tor left-censored results; the upper bound assigns the value of LOD or LOQ to
results below the LOD and LOQ, respectively.
The highest concentrations for AOH, AME, TeA and TEN were found in the food group Legumes,
nuts and oilseeds and in particular in sunflower seeds. Mean concentrations of AOH in this food
group were in the range of 22 g/kg (LB mean) to 26 g/kg (UB mean) with a maximum value of
1200 g/kg. For AME the mean values were in the range 11 (LB) to 12 g/kg (UB), with a maximum
value of 440 g/kg. TeA was present in higher concentrations (LB mean = 333 g/kg; UB
mean = 349 g/kg; maximum = 5400 g/kg). Mean concentrations of TEN ranged from 47 (LB mean)
to 50 g/kg (UB mean) with a maximum value of 880 g/kg.
Overall based on published occurrence data on about 300 feed and agricultural commodities in
Europe, AOH was found in 31 % of the feed and agricultural commodity samples at concentrations
from 6.3 to 1840 g/kg (maximum found in sunflower seeds). AME was found in 6 % of the samples
with levels ranging from 3 to 184 g/kg (maximum found in cereals). ALT was found in 73 % of the
samples with concentrations between 6.3 and 41 g/kg (maximum found in wheat grains). TeA was
present in 15 % of the samples with levels varying between 500 and 4310 g/kg (maximum found in
oats).
Scarce information is available on the behaviour of Alternaria toxins in food and feed during the
storage and processing but there are some indications that Alternaria toxin concentrations may
increase under favourable conditions and may be stable during food and feed processing.
Considering the limited occurrence data available and the high proportion of data below LOD/LOQ
(left-censored data), the CONTAM Panel decided to perform a limited dietary exposure assessment
focussing only on adults ( 18 to < 65 years old).
Although the chronic dietary exposure was not calculated for all age classes, due to the higher food
consumption per kg body weight (b.w.) it is expected that the dietary exposure in children might be
higher compared to adults by a factor of 2 to 3. Similarly, vegetarians might have higher exposure due
to the higher intake of food of plant origin.
The dietary exposure in adults was estimated only for AOH, AME. TeA and TEN where the
quantified results accounted for 7.7 %, 7.0 %, 13 % and 6.0 % of data, respectively. Due to the
absence or very limited number of quantified results for ALT (0 %), ATX-I (0.2 %) and AAL-toxins
(0 %), dietary exposure assessment was not performed for these toxins. Given the above limitations in
the occurrence data, the exposure estimate should be regarded as being only indicative.
The estimated mean chronic dietary exposure in the adult population across dietary surveys, using LB
and UB mean concentrations, was in the following ranges: AOH: 1.9 - 39 ng/kg b.w. per day; AME:
0.8 - 4.7 ng/kg b.w. per day; TeA: 36 - 141 ng/kg b.w. per day; TEN 0.01 - 7 ng/kg b.w. per day (the
ranges represent the minimum LB to maximum UB from the different countries). The 95th percentile
exposure estimates were 2 to 3 times higher compared to the mean dietary exposure estimates.
Depending on the Alternaria toxins and the food consumption pattern in the European countries,
based on the few available data, the contribution to the dietary exposure to AOH, AME, TeA and
EFSA Journal 2011;9(10):2407
TEN is mainly made by grain and grain-based products, vegetables and vegetable products in
particular tomato products, fruits and fruit products including fruit and vegetable juices, alcoholic
beverages (wine and beer), oilseeds and vegetable oils (mainly sunflower seeds and sunflower oil).
Estimation of intake of Alternaria toxins in feed by farm livestock was limited to chicken, because it
was the only species for which some toxicity data suitable for risk assessment exist. Since the
occurrence data on feed were insufficient for most of the Alternaria toxins, the exposure estimates
were limited to AOH. The calculated LB and UB exposures to AOH are about 3 g/day and about
6 g/day, respectively, for both broilers and laying hens. The exposure estimate should be regarded as
being only indicative.
The information on metabolism of Alternaria toxins in the mammalian organism is limited to AOH,
AME and ALT. Experiments carried out in vitro indicate that they are hydroxylated mostly to
catechol metabolites and conjugated with glucuronic acid and sulphate. There is no relevant
information available on the absorption, distribution and excretion of any Alternaria toxin in animals
and humans.
AOH and AME have been reported to be genotoxic in bacteria and mammalian cells in vitro. ATXs
are mutagenic in bacteria and induce cell transformation, while TEN and TeA are not mutagenic in
bacteria. There are no in vivo genotoxicity or carcinogenicity data available for Alternaria toxins.
Some indications of precancerous changes have been reported in oesophageal mucosa of mice.
Acute oral toxicity of TeA has been studied in several animal species (LD50 37.5 and 225 mg/kg b.w.,
for chicks and mice, respectively). Reproductive and developmental studies are limited and no toxic
effects from oral administration of Alternaria toxins have been reported. Data on sensitivity of farm
and companion animals towards Alternaria toxins are very limited and do not allow the estimation of
tolerance levels for individual toxins and mixtures thereof.
The database concerning toxicological effects of Alternaria toxins in experimental animals and/or in
humans is currently too limited to be used for identification of reference points for different
toxicological effects. Experiments performed in rodents with purified Alternaria toxins indicate that
the acute toxicity is in the following order: ALT > TeA > AME and AOH. However, these data are
not suitable for the risk assessment of Alternaria toxins since the risk for public health related to these
toxins is not expected to result from acute exposures. The available short term toxicity studies were
not suitable for risk assessment purposes.
Considering that there are few or no relevant toxicity data on Alternaria toxins, and that the chemical
structure of several of them is known, the CONTAM Panel considered it appropriate to use the
threshold of toxicological concern (TTC) approach to assess the relative level of concern of these
mycotoxins for human health. The CONTAM Panel considered that the occurrence data of AOH,
AME, TeA and TEN were adequate to apply the TTC approach and therefore based the assessment on
the mean and 95th percentile chronic dietary exposure to AOH, AME, TeA and TEN for the adult
population using LB and UB. For the genotoxic Alternaria toxins, AOH and AME, the estimated
mean chronic dietary exposures at the UB and 95th percentile dietary exposures exceed the TTC value
of 2.5 ng/kg b.w. per day, indicating a need for additional compound-specific toxicity data. TeA and
TEN were negative in bacterial mutagenicity assays. The TTC value for this type of non-genotoxic
substances is 1500 ng/kg b.w. per day. For TEN, the estimated mean chronic dietary exposures at the
UB and the 95th percentile dietary exposures are more than 4-fold lower than the TTC value,
indicating that TEN is considered unlikely to be of a human health concern. Estimates of the chronic
dietary exposure to TeA ( 13 ng/kg b.w. per day) are much lower than the TTC value and is
therefore considered unlikely to be a human health concern
At present, the knowledge on the possible effects of Alternaria toxins on farm and companion animals
as well as the database describing the occurrence of these mycotoxins in feedstuffs are scarce and are
not sufficient to assess the risk regarding Alternaria toxins for animal health. The estimation of the
EFSA Journal 2011;9(10):2407
intake of AOH by chickens on the basis of UB values was found to be about 6 g/day. Since no
evidence of toxicity was observed in chicks at approximately 17-fold higher levels, it is unlikely that
AOH represents a health risk for broilers or laying hens at the present estimated level of intake.
Regarding TeA, assuming that chicken could be fed exclusively with wheat this would result in a TeA
intake of about 120 g/day, corresponding to approximately 5 % of the lowest-observed-adverseeffect level. This estimation suggests that adverse health effects of feed containing TeA cannot fully
be ruled out in chickens. The lack of toxicological data precludes any conclusions for other species.
EFSA Journal 2011;9(10):2407
TABLE OF CONTENTS
Abstract .................................................................................................................................................... 1
Summary .................................................................................................................................................. 2
Table of contents ...................................................................................................................................... 6
Background as provided by the European Commission........................................................................... 8
Terms of reference as provided by the European Commission ................................................................ 9
Assessment ............................................................................................................................................. 10
1. Introduction ................................................................................................................................... 10
1.1.
Alternaria toxin production .................................................................................................. 10
1.2.
Previous assessments ............................................................................................................ 12
1.3.
Chemistry and physical properties ........................................................................................ 12
2. Sampling and methods of analysis ................................................................................................ 16
2.1.
Sampling ............................................................................................................................... 16
2.2.
Methods of analysis of Alternaria toxins in food and feed................................................... 16
2.2.1. Sample preparation ........................................................................................................... 16
2.2.2. Instrumental techniques .................................................................................................... 17
2.2.2.1. Chromatographic techniques ................................................................................... 17
2.2.2.2. Immunochemical methods ....................................................................................... 18
2.2.2.3. Reference materials, validation and proficiency testing .......................................... 18
3. Occurrence of Alternaria toxins in food and feed ......................................................................... 18
3.1.
Previously reported occurrence results of Alternaria toxins in food .................................... 18
3.2.
Currently reported occurrence results of Alternaria toxins in food ...................................... 20
3.2.1. Data collection summary .................................................................................................. 20
3.2.2. Distribution of samples across food groups...................................................................... 21
3.2.3. Analytical methods used ................................................................................................... 22
3.2.4. Occurrence data by food category .................................................................................... 23
3.3.
Previously reported occurrence results of Alternaria toxins in feed..................................... 29
3.4.
Occurrence data in feed......................................................................................................... 31
3.5.
Trends in the previously reported occurrence data ............................................................... 34
3.6.
Storage and processing of food and feed .............................................................................. 36
4. Food and feed consumption ........................................................................................................... 37
4.1.
Food consumption................................................................................................................. 37
4.1.1. EFSA Comprehensive European Food Consumption Database ....................................... 37
4.2.
Feed consumption ................................................................................................................. 38
5. Exposure assessment of Alternaria toxins in animals and humans ............................................... 39
5.1.
Human exposure assessment ................................................................................................. 39
5.1.1. Mean and high dietary exposure to Alternaria toxins ...................................................... 39
5.1.1.1. Contributions of different food groups to exposure of Alternaria toxins ................ 41
5.2.
Estimation of intake Alternaria toxins in feed by farm livestock ......................................... 42
6. Hazard identification and characterisation .................................................................................... 43
6.1.
Toxicokinetics: animal and in vitro studies .......................................................................... 43
6.2.
In vitro toxicity ..................................................................................................................... 44
6.2.1. Cytotoxicity ...................................................................................................................... 44
6.2.1.1. Mechanism of cytotoxicity ...................................................................................... 45
6.2.2. Genotoxicity ..................................................................................................................... 45
6.2.2.1. Bacterial cells ........................................................................................................... 45
6.2.2.2. Mammalian cells ...................................................................................................... 46
6.2.2.3. Mechanism of genotoxicity...................................................................................... 46
6.3.
Toxicity in animals ............................................................................................................... 47
6.3.1. Laboratory animals and bioassays .................................................................................... 47
6.3.1.1. Acute toxicity ........................................................................................................... 47
6.3.1.2. Short term toxicity ................................................................................................... 48
EFSA Journal 2011;9(10):2407
6.3.1.3. Reproductive and developmental toxicity ............................................................... 49

6.3.1.4. Mechanism of reproductive and developmental toxicity ......................................... 49
6.3.1.5. Carcinogenicity ........................................................................................................ 50
6.3.2. Farm and companion animals ........................................................................................... 50
7. Risk characterisation...................................................................................................................... 51
7.1.
Human health risk assessment .............................................................................................. 51
7.2.
Animal health risk assessment .............................................................................................. 52
8. Uncertainty analysis ...................................................................................................................... 52
8.1.
Assessment objectives........................................................................................................... 53
8.2.
Exposure scenario and exposure model ................................................................................ 53
8.3.
Model input (parameters) ...................................................................................................... 53
8.4.
Other uncertainties ................................................................................................................ 53
8.5.
Summary of uncertainties ..................................................................................................... 53
Conclusions and recommendations ........................................................................................................ 54
References .............................................................................................................................................. 58
Appendices ............................................................................................................................................. 70
Abbreviations ......................................................................................................................................... 96
EFSA Journal 2011;9(10):2407
BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION

Alternaria is a common genus of fungi, and contains numerous species that are both saprophytic on
organic materials and pathogenic on many plants. A number of species can contaminate a wide variety
of crops in the field and cause post-harvest decay of various fruits, grains, and vegetables causing
considerable losses due to rotting of fruits and vegetables.
In addition to causing economic losses, Alternaria sp. form under certain conditions mycotoxins with
toxic properties. As such, the occurrence of Alternaria mycotoxins in foodstuffs such as grains, peanuts,
tomato products, apple sauce, olive oil, fresh fruits and vegetables, is becoming an increasing concern
for public health.
The Alternaria genus produces more than 70 known mycotoxins and phytotoxins but only a few occur
in food or are of major toxicological significance. A. alternata is probably the most important
mycotoxin producing species and occurs e.g. on cereals, sunflower seeds, oilseed rape, olives and
various fruits.
Alternaria toxins which contaminate food are (not exhaustive): alternariol, alternariol monomethyl
ether, tentoxin, tenuazonic acid, altertoxins, stemphyltoxin III, Alternaria alternata f. sp.lycopersici
toxins. The table hereafter provides a more complete overview.
Table 1:
Not exhaustive list of Alternaria toxins and producing Alternaria species.
Mycotoxin
Alternariol
Alternariol monomethyl ether
Tentoxin
Tenuazonic acid
Altertoxins
Alternaria
alternata
f.
lycopersici toxins
Stemphyltoxin III
Altenuene
sp
Acronym
AOH
AME
TEN
TeA
ATXs
AAL-toxins
Species Producing
Alternaria alternata
A. alternata; A. solani
A. alternata
A. alternata; A. tenuissima
A. alternata.
A. alternata f. sp. lycopersici
Stemphyltoxin III
ALT
A. alternata
A. alternata
Available information (not exhaustive)

In accordance with Article 36 of Regulation (EC) No 178/2002, a report Scientific information on
mycotoxins and natural plant toxicants has been produced following a grant agreement between the
European
Food
Safety
Authority
(EFSA)
and
the
author(s)
of
the
report
(CFP/EFSA/CONTAM/2008/01). The report presents information, inter alia, regarding Alternaria
mycotoxins in feed and food and is available on the EFSA website4.
An EU-funded research project has been carried out in the period from 2000 to 2004 out on: Safe
organic vegetables and vegetable products by reducing risk factors and sources of fungal contaminants
throughout the production chain: the carrot Alternaria model. The project aimed at monitoring and
reducing the mycotoxin contamination in the production chain of carrots, and products derived from
them, and focused on:
1. Establishing rapid and precise methods for detection and quantification of Alternaria and
Alternaria mycotoxins in carrot material
http://www.efsa.europa.eu/en/scdocs/doc/24e.pdf
EFSA Journal 2011;9(10):2407
2. Establishing the basic understanding of Alternaria mycotoxin production and accumulation in

the production chain to determine critical control points and to enable risk assessment
3.
Developing control measures, acceptable to organic farmers, to prevent the introduction of

Alternaria and mycotoxin accumulation in the carrot production chain
4. Developing a total strategy to reduce the risk of mycotoxins in organic carrots and products
derived from them, and to extrapolate the results to other organic vegetables where possible
The outcome of the research project is made available to EFSA.
Issue
There might be a possible risk for animal and public health related to the presence of Alternaria toxins
in feed and food. The European Commission asks EFSA to assess on the basis the available information
the risk for animal and public health in order to enable the European Commission and the competent
authorities in the Member States to consider the need for a possible follow up including to fill the
knowledge gaps.
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 to provide a scientific opinion on the risks for public health related to
the presence of Alternaria toxins in feed and food.
The assessment should, based upon the available information, assess if the presence of Alternaria toxins
in food and feed is a potential risk for public and animal health taking into account the toxicity of the
different Alternaria toxins and their occurrence in feed and food. For the assessment of the risks, the
situation for the different animal species and the specific (vulnerable) groups of the human population
(e.g. high consumers, children, people following specific diets, etc.) should be considered.
EFSA Journal 2011;9(10):2407
ASSESSMENT
1.
Introduction
Alternaria species are fungi widely distributed in the soil as normal components of its microflora and
are both saprophytes and plant pathogens. They are widespread in both humid and semi-arid regions and
can infect growing plants in the field. Alternaria species cause plant diseases on many crops, affecting
the leaves, stems, flowers, and fruits. They are the principal contaminating fungi in wheat, sorghum and
barley (Deshpande, 2002). In addition to cereal crops, Alternaria species have been reported to occur in
oilseeds such as sunflower and rape seed, tomato, apples, citrus fruits, olives and several other fruits and
vegetables. Many Alternaria species are host-specific; for example Alternaria alternata f. sp.
lycopersici Keissl. causes necrotic lesions in tomato. Due to their growth even at low temperature,
Alternaria species are also responsible for spoilage of these commodities during refrigerated transport
and storage.
Alternaria species produce more than 70 secondary metabolites which are toxic to plants. A small
proportion of these phytotoxins have been chemically characterised and reported to act as mycotoxins to
humans and animals (Bottalico and Logrieco, 1992; Barkai-Golan, 2008) A. alternata (Fries) Keissl.
which is the most common Alternaria species in harvested fruits and vegetables, is the most important
mycotoxin-producing species. Based on their effect on plants, Alternaria toxins are divided into nonhost-specific toxins and host-specific toxins. Some non-host-specific toxins such as alternariol,
alternariol monomethyl ether, tenuazonic acid and altertoxins have been tested individually and are
described to induce harmful effects in animals, including fetotoxic and teratogenic effects. Culture
extracts of A. alternata as well as individual mycotoxins such as alternariol and alternariol monomethyl
ether are mutagenic and genotoxic in various in vitro systems. In addition, it has been suggested that in
certain areas in China Alternaria toxins in grains might be responsible for oesophageal cancer but the
presence of other toxigenic fungi in these grains renders this association somewhat inconclusive. On the
other hand, the animal toxicity of host-specific toxins such as A. alternata f. sp. lycopersici toxins
(AAL-toxins) have not been fully examined. Hence, due to their possible harmful effects, Alternaria
toxins are of concern for public health. They have not been reported to cause animal toxicosis as a result
of exposure from feed. This opinion deals with alternariol, alternariol monomethyl ether, tenuazonic
acid, iso-tenuazonic acid, altertoxins, tentoxin and AAL-toxins. However, several other Alternaria
toxins have been identified. Alternaria spores are known to be allergenic via inhalation and Alternaria
skin and nasal infections can occur in immunocompromised patients. However, since no evidence exists
that these pathologies are related to Alternaria toxins, these toxicological endpoints are not considered
in this opinion. Currently there are no regulations on Alternaria toxins in food and feed in Europe or in
other regions of the world.
1.1.
Alternaria toxin production
Alternaria species are fungal necrotrophs commonly present on dead organic matter. Their spores are
dispersed by air currents and are considered among the most important fungal aeroallergens (Budd,
1986). Alternaria causes plant diseases of economic importance on many crops in the European Union
(EU) and worldwide, affecting the leaves, stems, flowers, and fruits (Appendix A, Table A1). Alternaria
diseases are characterised by a great environmental adaptation, inducing severe epidemics in both humid
and semi-arid regions. Some Alternaria species are able to infect and induce symptoms on plants during
their growing stages, while others only cause damage after harvest in storage, trade and processing.
Total losses caused by this genus rank among the highest caused by any plant pathogen (Agrios, 2005).
Species of Alternaria were traditionally defined based on spore characteristics and sporulation patterns
(Simmons, 1992). The genus was divided into several morphological species-groups, which were
EFSA Journal 2011;9(10):2407
10
further considered as phylogenetic groups in molecular and metabolite profiling studies (Pryor and
Gilbertson, 2000; Andersen et al., 2002; Pryor and Bigelow, 2003; Hong et al., 2006; Andersen et al.,
2008). Phylogenetic studies have demonstrated a clear distinction between large and small-spored
Alternaria species, but boundaries among the small-spored taxa are still a subject of controversy (Pryor
and Bigelow, 2003; Peever et al., 2004; Andrew et al., 2009).
Spores of Alternaria have melanized walls, which protect them from the ultraviolet (UV) radiation and
desiccation. Spores have a high infection efficiency due to the production of several germ tubes during
the germination process. Environmental requirements for infection depend on the Alternaria species and
the host, but the interaction of adequate temperature and a film of water produced by rain, dew
condensation or overhead irrigation is always necessary. Optimum temperature for mycelial growth is
around 18-25C, but spores are able to germinate and infect in a range of 4 to 35C. Under optimal
temperature conditions, at least 5-8 hours of wetness are required for infection (Rotem, 2004; BarkaiGolan, 2008).
After infection, Alternaria species require a relative high water content (> 20 %) to colonize plant
tissues. Thus, Alternaria rots are more common in fruits and vegetables than in seeds and grains, which
are usually stored at low moisture. However, Alternaria growth may be favoured in grains harvested
during wet weather and stored in moist conditions (Bottalico and Logrieco, 1998). Alternaria species
are capable of growing during refrigerated storage, but they may also cause latent infections which
remain dormant until conditions become more favourable for fungal growth. During storage, Alternaria
may also spread from affected plant products to adjacent healthy ones by secondary infections (Rotem,
1994; Barkai-Golan, 2008).
Alternaria sporulate on necrotic and dead plant tissues. Some species have a diurnal pattern of
sporulation, where light induces the formation of spore-bearing structures and the production of spores
is enhanced by darkness. Optimum temperatures for sporulation of different species range from 15 to
30C. Moisture is also necessary for sporulation but, in contrast to infection, relative humidities below
saturation may be sufficient. Spore release is triggered by rain events and sudden changes in relative
humidity. Alternaria spores are disseminated mainly by air currents, but they can be also rain splashed
(Rotem, 1994). Once released, Alternaria spores are able to withstand adverse environmental conditions
for several days, and conclude infection when weather becomes favourable. Alternaria is also well
adapted for long-term survival and overseasoning. Fungal propagules in affected seeds and plant debris
have a longevity of several years (Rotem, 1994).
Alternaria species produce extracellular enzymes that degrade plant cell wall polymers during infection.
Most species also produce phytotoxins (plant toxins) which act in different sites causing cell death in
plant tissues (Appendix A, Table A2 ). Based on their effect on plants, these toxins are divided into nonhost-specific toxins and host-specific toxins (Thomma, 2003). Generally, non-host-specific toxins such
as tenuazonic acid, tentoxin and zinniol, affect a broad range of plant species and are considered as
virulence factors rather than key pathogenicity determinants. However, the mode of action of most of
these toxins in plant infection remains unstudied. Some non-host-specific toxins such as alternariol,
alternariol monomethyl ether, tenuazonic acid and altertoxins have been also described to induce
potentially harmful effects in mammals (Barkai-Golan, 2008). On the other hand, host-specific toxins
such as AAL-toxins have a limited host range and play a critical role in plant pathogenicity, but their
animal toxicities have not been fully examined (Thomma, 2003; Barkai-Golan, 2008).
Toxin production is affected by interactions among Alternaria strain, the growing substrate and the
environmental conditions (Barkai-Golan, 2008). The production of non-host specific toxins on
standardized laboratory media has been utilized to distinguish between morphological species-groups in
Alternaria. Several pathotypes of the species A. alternata have been also defined based on the presence
of host-specific toxins (Andersen et al., 2001, 2002; Thomma, 2003; Andersen et al., 2008; Somma et
al., 2011). However, due to the different taxomic criteria used in the literature, it is difficult to establish
precise relationships between Alternaria species and toxin production. Alternaria can grow and produce
EFSA Journal 2011;9(10):2407
11
toxins both on artificial and natural substrates as long as moisture content is above 25-30 %. The
optimum pH for mycelial growth is between 4 and 6.6, but Alternaria tolerates a range of 2.7 to 8. No
growth was observed in absence of oxygen (Bottalico and Logriego, 1998). Temperatures for toxin
production are similar to those described for infection and mycelial growth and sporulation. However,
significant amounts of toxins were detected even at suboptimal temperatures (Ozcelik et al., 1990;
Hasan, 1995; Barkai-Golan, 2008; Oviedo et al., 2009). Therefore, foods of plant origin which are
refrigerated during the transportation and storage may be contaminated (Ozcelik et al., 1990; Vias et
al., 1992).
Fresh plant products affected by Alternaria regularly present visible rotten areas, which are usually
removed or avoided by the consumer (Logriego et al., 2009). However, Alternaria toxins can be
transferred from the rotten part to the surrounding tissues (Robiglio and Lopez, 1995). This may be
particularly relevant for Alternaria diseases whose symptoms are present only in the internal part of the
affected organ, such as core rot of apples and black rot of citrus. Toxins are also present in processed
plant products due to the limitations of the current industrial procedures to completely eliminate the
rotting tissues (Logriego et al., 2009). Some crops affected by Alternaria diseases, such as grains, pome
fruits and grapes, are often infected also by toxigenic strains of Fusarium, Penicillium and Aspergillus,
so the co-occurrence of Alternaria toxins with other mycotoxins is likely to occur (Agrios, 2005;
Weidenbrner, 2008).
1.2.
Previous assessments
There are no previous risk assesments on Alternaria toxins in food and feed carried out at the European
or at international level. A few brief national assessments were only identified from Europe. In 2003,
the Federal Institute of Risk Assessment (BfR) in Germany considered Alternaria toxins and concluded
that the data are currently not sufficient to carry out a risk assessment on the presence of Alternaria
toxins in food and feed, and that more occurrence and toxicological data are needed (BfR, 2003). The
risk assessment conducted in France concluded that currently Alternaria toxins in food or feed are not
considered as priority toxins regarding a risk for human or animal health (AFSSA, 2009). Nevertheless
because toxicological studies, essentially conducted in vitro, indicate mutagenic effects, a
recommendation was given to perform in vivo genotoxicity studies in animals receiving doses
compatible with existing contamination data (AFSSA, 2009). The Scientific Committee on Food of the
Czech Republic could not carry out an exposure assessment but it recommended to continue to study
genotoxicity and carcinogenicity of Alternaria toxins (e.g. alternariol and alternariol monomethyl ether)
and to conduct additional relevant toxicity studies on Alternaria toxins such as alternariol, alternariol
monomethyl ether, altenuene, tenuazonic acid, and altertoxins I, II and III (Czech SCF, 2008). It appears
that risk assessments on Alternaria toxins in food or feed have not been conducted outside Europe.
1.3.
Chemistry and physical properties
Alternaria toxins are divided into 5 different classes based on their chemical structures: (1) dibenzo-pyrones which include alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT);
(2) tenuazonic acid (TeA) and iso-tenuazonic acid (iso-TeA); (3), perylene quinones which include
altertoxins I, II and III (ATX-I, ATX-II and ATX-III), and stemphyltoxin III; (4) AAL-toxins,
abbreviation for A. alternata f. sp. lycopersici toxins. AAL-toxins include 2 groups, AAL-TA and AALTB. AAL-TA consists of two esters (C13 or C14) of 1,2,3-propane-tricarboxylic acid and 1-amino11,15-dimethylheptadeca-2,4,5,13,14 pentol. AAL-TB consists of two esters at C13 or C14 of
1,2,3-propane-tricarboxylic acid and 1-amino-11,15-dimethylheptadeca-2,4,13,14-tetrol. The 5th class
contains miscellaneous structures such as tentoxin (TEN) (a cyclic tetrapeptide). The chemical
structures and names of the most studied Alternaria toxins are presented in Figures 1 and 2 and Table 2.
EFSA Journal 2011;9(10):2407
12

OH
2
H3 C
H3 C
10
HO
OH
H3 C
8
7
O 5
OH
AOH
H3 C
O 5
OH
10
CH3
O 5
OH
AME
OH
10
OH
ALT
HO
HO
CH3
CH3
H3 C
N
CH3
H3 C
CH3
TeA
iso-TeA
O
O
OH
H
HO
OH
O
OH
O
HO
H
OH
HO
ATX-I
ATX-II
OH
O
O
H
OH
OH
H
HO
HO
O
O
ATX-III
Stemphyltoxin III
Figure 1: Chemical structures of AOH, AME, ALT, stemphyltoxin III, ATX-I, ATX-II, ATX-III,
TEN, TeA and iso-TeA.
EFSA Journal 2011;9(10):2407
13
COOH
HOOC
CH3
14
CH3
OH
NH2
13
OH
OH
OH
AAL TA1
CH3
OH
14
13
CH3
OH
NH2
OH
OH
AAL TA2
COOH
COOH
HOOC
COOH
CH3
O
14
CH3
NH2
13
OH
OH
OH
AAL TB1
CH3
CH3
OH
14
NH2
O
CH3
13
OH
OH
NH
AAL TB2
COOH
N
CH3
CH 3
O
H
NH
H
CH3
CH 3
COOH
TEN
Figure 2: Chemical structures of AAL toxins.
EFSA Journal 2011;9(10):2407
14
Table 2: Chemical names, Chemical Abstracts Services (CAS) Registry Numbers, molecular weights
and molecular formulas of AOH, AME, ALT, ATX-I, ATX-II, ATX-III, TeA,TEN, stemphyltoxin III,
and AAL-TA1, AAL-TA2, AAL-TB1 and AAL-TB2.
Chemical name
Toxin/
Common
name
AOH/
3,7,9-Trihydroxy-1-methyl-6H-dibenzo[b,d]pyran-6-one
Alternariol
3,7-Dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one
AME/
Alternariol
monomethyl
ether
ALT/
(2R,3R,4aR)-rel-2,3,4,4a-Tetrahydro-2,3,7-trihydroxy-9-methoxyAltenuene
4a-methyl-6H-dibenzo[b,d]pyran-6-one
ATX-I/
(1S,12aR,12bS)-1,2,11,12,12a,12b-Hexahydro-1,4,9,12aAltertoxin-I
tetrahydroxy-3,10-perylenedione
ATX-II
(7aR,8aR,8bS,8cR)-7a,8a,8b,8c,9,10-Hexahydro-1,6,8c-trihydroxyAltertoxin-II
perylo[1,2-b]oxirene-7,11-dione
ATX-III
(1aR,1bS,5aR,6aR,6bS,10aR)-1a,1b,5a,6a,6b,10a-Hexahydro-4,9Altertoxin-III
dihydroxy-perylo[1,2-b:7,8-b']bisoxirene-5,10-dione
(5S)-3-Acetyl-1,5-dihydro-4-hydroxy-5-[(1S)-1-methylpropyl]-2HTeA/
pyrrol-2-one
Tenuazonic
acid
TEN/
Cyclo[N-methyl-L-alanyl-L-leucyl-(Z)-,-didehydro-NTentoxin
methylphenylalanylglycyl]
Stemphyl(7aR,8aR,8bS,8cR)-7a,8a,8b,8c-Tetrahydro-1,6,8c-trihydroxytoxin III
peryleno[1,2-b]oxirene-7,11-dione
(2R)-1,2,3-Propanetricarboxylic acid, 1-[(1S,3S,9R,10S,12S)-13AAL-TA1/
A. alternata f. amino-9,10,12-trihydroxy-1-[(1R,2R)-1-hydroxy-2-methylbutyl]-3sp. lycopersici methyltridecyl] ester
toxins TA1
(2R)-1,2,3-Propanetricarboxylic acid, 1-[(1R,2S,4S,10R,11S,13S)AAL-TA2/
A. alternata f. 14-amino-2,10,11,13-tetrahydroxy-4-methyl-1-[(1R)-1sp. lycopersici methylpropyl]tetradecyl] ester
toxins TA2
(2R)-1,2,3-Propanetricarboxylic acid, 1-[(1S,3S,10R,12S)-13AAL-TB1/
A. alternata f. amino-10,12-dihydroxy-1-[(1R,2R)-1-hydroxy-2-methylbutyl]-3sp. lycopersici methyltridecyl] ester
toxins TB1
(2R)-1,2,3-Propanetricarboxylic acid, 1-[(1R,2S,4S,11R,13S)-14AAL-TB2/
A. alternata f. amino-2,11,13-trihydroxy-4-methyl-1-[(1R)-1sp. lycopersici methylpropyl]tetradecyl] ester
toxins TB2
CAS No: Chemical Abstracts Services Registry Number; MW: molecular weight.
CAS No
MW
(Da)
Molecular
formula
641-38-3
258
C14H10O5
23452-05-3
272
C15H12O5
29752-43-0
292
C15H16O6
56258-32-3
352
C20H16O6
56257-59-1
350
C20H1406
105579-74-6
348
C20H12O6
610-88-8
197
C10H15O3N
28540-82-1
414
C22H30O4N4
102694-32-6
348
C20H12O6
79367-52-5
521
C25H47O10N
79367-51-4
521
C25H47O10N
176590-32-2
505
C25H47O9N
176705-51-4
505
C25H47O9N
Physico-chemical characteristics are reported in the literature only for some of the Alternaria toxins. In
particular, TeA is a colourless, viscous oil and is a monobasic acid with pKa 3.5. It is soluble in
methanol and chloroform. On standing, heating or treatment with a base, optical activity is lost and
crystallisation may occur as a result of the formation of iso-TeA. It forms complexes with calcium,
magnesium, copper, iron and nickel ions.
AOH and AME crystallise from ethanol as colourless needles, and melting points with decomposition
are 350C and 267C, respectively. They are soluble in most organic solvents and give a purple colour
reaction with ethanolic ferric chloride. ALT crystallises as colourless prisms melting at 190-191C.
ATX-I is an amorphous solid melting at 180C, which shows a characteristic bright yellow fluorescence
under ultraviolet (UV) light. Yellow-orange fluorescence is characteristic for ATX and violet-blue for
AOH, AME and ALT (Betina, 1993).
EFSA Journal 2011;9(10):2407
15
The first Alternaria toxins were isolated, characterised and confirmed in the years 1953-1986. To
provide sufficient amounts of material for toxicological and biological testing, total syntheses of AOH,
AME and ALT have been established (Koch et al., 2005; Altemller et al., 2006). Alternaria toxins can
be partly metabolised in plants, e.g. the formation of conjugated metabolites (Berthiller et al., 2005).
These modified or "masked" mycotoxins, in which the toxin is usually bound to a more polar substance
such as glucose, amino acids and sulfates are of concern as they probably can release their native
precursors after enzymatic hydrolysis in the digestive tract of organisms. They are referred to as masked
mycotoxins because they have the ability to escape established analytical methods due to differences in
polarity between the native precursors and their metabolites. Research has mainly focused on the parent
mycotoxins while very limited data are available on the occurrence of mycotoxin metabolites in food
and feed.
The chemical stability of AOH, AME and ALT has been studied by Siegel et al. (2010a) by refluxing
the toxins in aqueous solutions with different pH for 5 hours. All three compounds were stable in
0.15 M phosphate buffer pH 5, but were completely degraded in 0.1 M KOH to undefined brown
products. ALT was stable in 0.18 M phosphate/citrate buffer pH 7, but AOH and AME were degraded
to 6-methylbiphenyl-2,3,4,5-tetrol and 5-methoxy-6-methylbiphenyl-2,3,4-triol, respectively. The
mechanism of degradation is thought to involve hydrolysis of the lactone group followed by
decarboxylation.
2.
Sampling and methods of analysis
2.1.
Sampling
Sampling for the official control of the levels of mycotoxins has been laid down in the Commission
Regulation (EC) No 401/2006 of 23 February 2006.5 However, the regulation only concerns aflatoxin
B1, total aflatoxins, ochratoxin A and Fusarium-toxins in cereals and cereal products. Sampling
protocols for monitoring Alternaria toxins in food products e.g. carrots and tomatoes are not described
in the EU legislation.
2.2.
Methods of analysis of Alternaria toxins in food and feed
The methods of analysis of Alternaria toxins have been reviewed by Scott (2001) and more recently by
Ostry (2008), Shephard et al. (2009, 2010) and Kppen et al. (2010).
2.2.1.
Sample preparation
For the isolation of Alternaria toxins from food products either liquid/liquid extraction (LLE) or solid
phase extraction (SPE) techniques mainly based on C18, aminopropyl and hydrophilic-lipophilic
balance (HLB) columns are used. LLE is applied for tomatoes and tomato products (Scott and Kanhere,
1980; Stack et al., 1985; da Motta and Soares, 2000a,b Andersen and Frisvad, 2004) fruit juices and
beverages (Lau et al., 2003), oilseed rape meal and sunflower seed meal (Nawaz et al., 1997), cereal
samples (Siegel et al., 2009), beer (Siegel et al. 2010b), hay, silage and mixed feed (Yu et al., 1999),
wheat and maize (Herebian et al., 2009), maize silage (Rasmussen et al., 2010) and ground breadcrumbs
mouldy food samples (Sulyok et al., 2007, 2010). For the isolation of the Alternaria toxins from the
samples either organic solvents (methanol, ethyl acetate, chloroform) or mixtures of organic solvents
with (buffered) water are used.
Commission Regulation (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the
official control of the levels of mycotoxins in foodstuffs. OJ L 70, 9.3.2006, p. 12-70, and its amendment Commission
Regulation (EU) No 178/2010 of 2 March 2010, OJ L 52, 3.3.2010, p. 32-43.
EFSA Journal 2011;9(10):2407
16
SPE, after appropriate dilution of the sample, is used for cleaning up beverage samples (Asam et al.,
2009), apple juice (Delgado et al., 1996), tomato paste (Fente et al., 1998), fruit juices and beverages
(Lau et al., 2003), tangerines (Magnani et al., 2007) and apple juice (Scott et al., 1997).
The combination of LLE with SPE is applied for carrots (Solfrizzo et al., 2004), food supplements
(Diana di Mavungu et al., 2009), sweet peppers (Monbaliu et al., 2009), cereal, fruit and vegetable
products (Asam et al., 2010) and irradiated soya beans (Oviedo et al., 2011). Nawaz et al. (1997)
combined LLE with gel permeation chromatography (GPC) for the detection of Alternaria toxins in
oilseed rape meal and sunflower seed meal.
2.2.2.
Instrumental techniques
2.2.2.1. Chromatographic techniques

A few methods using thin-layer chromatography (TLC) have been reported (Matysik and Giryn, 1996;
Skarkova et al., 2005,). Fluorescence detection was used for AOH, AME and ALT, while spraying with
a solution of iron (III) chloride in ethanol, followed by UV examination at 254 nm was needed for TeA.
The limit of quantification (LOQ) of AOH, AME and ALT was 5 g/kg and of TeA 25 g/kg in rape
seed and wheat samples.
The use of gas chromatography (GC) for analysis of Alternaria toxins in food and feed matrices
requires pre-column derivatisation. Derivatisation to heptafluorobutyrates (HFB) and
trimethylsilylderivatives is appropriate for separation of Alternaria toxins by GC prior to detection by
electron impact (EI) mass spectrometry (Scott et al., 1997). A limit of detection (LOD) of 1 g/kg for
AOH and AME in apple juice was reported for the GC method (Scott et al., 1997).
High performance liquid chromatography (HPLC) methods with fluorescence detection (FLD) (Stack et
al., 1985, Nawaz et al., 1997, Fente et al., 1998, Andersen and Frisvad, 2004), UV-detection (Scott and
Kanhere. 1980, Stack et al. 1985, Nawaz et al., 1997; Oviedo et al., 2011) and diode array detection
(DAD) (Delgado et al., 1996; da Motta and Soares, 2000a,b; Andersen and Frisvad, 2004; Solfrizzo et
al. 2004) have also been reported for food and feed. The LODs ranged from 0.7 g/kg for AME in apple
juice (Delgado et al., 1996) to 350 g/kg for TeA in sunflower seed meal (Nawaz et al., 1997). LOQ of
2 g/kg for AME and 5 g/kg for AOH in tomato products were reported (da Motta and Soares,
2000a,b).
Evaporative light-scattering detection has been coupled to HPLC for the detection and quantification of
A. alternata f.sp. lycopersici toxins in cultures (Li and Liangchen, 2006). Alternaria toxins in cultures
of Alternaria toxins producing strains have also been analysed by various (semi) preparative techniques.
Such is the case for the analysis of ATX-I by high-speed counter-current chromatography (Hu et al.,
2008) and of metabolites produced by the Alternaria genus by TLC (Fbrega et al., 2002).
Most of the recent analytical methods used for the analysis of Alternaria toxins include reversed phase
HPLC or ultra performance liquid chromatography (UPLC) coupled to single MS or tandem MS
(MS/MS) (Scott, 2001; Lau et al., 2003; Magnani et al., 2007; Sulyok et al., 2007; Asam et al., 2009;
Diana Di Mavungu et al., 2009; Herebian et al., 2009; Monbaliu et al., 2009; Siegel et al., 2009,
2010a,b; Asam et al., 2010; Rasmussen et al., 2010; Sulyok et al., 2010). LODs of 1 g/kg for AOH,
AME and ALT in maize and wheat (triple quadrupole technology) and in the range of 100 to 200 g/kg
in the same materials (ion trap technology), were reported by Herebian et al. (2009). In sweet peppers
LOD and LOQ for AOH, AME and ALT were 0.6-17 g/kg and 1.2-34 g/kg, respectively (Monbaliu
et al., 2009). LOD and LOQ for the same toxins in herbal medicines or food supplements (soy
isoflavones, St Johns Worth, garlic, Ginko biloba and black radish) were in the range of 2-30 g/kg
and 6-100 g/kg, respectively (Diana Di Mavungu et al., 2009).
EFSA Journal 2011;9(10):2407
17
2.2.2.2. Immunochemical methods

Direct competitive enzyme-linked immunosorbent assays (ELISA) have been used for the analysis of
AAL-TA in hay, silage and mixed feed with a LOQ of 50 g/kg (Yu et al., 1999). ELISA using
polyclonal rabbit antibodies has recently been reported for AOH and applied to contaminated maize and
animal feed with a LOQ of 20 g/kg (Burkin and Kononenko, 2011). Likewise, polyclonal rabbit
antibodies and monoclonal mouse antibodies have been generated against AOH and implemented in an
enzyme immunoassay (EIA) with a LOD of 1-2 g/kg (Ackermann et al., 2011).
No immunochemical methods have yet been developed for AME, ALT, ATX-I and TeA in foodstuffs
(Ostry 2008).
2.2.2.3. Reference materials, validation and proficiency testing
Solid standards for AOH, AME, ALT, ATX-I, TEN and TeA copper salt are commercially available.
However, these standards have not been officially certified. In contrast, certified standard solutions of
AOH, AME and TEN are commercially available. Certified reference materials for Alternaria toxins are
not available.
Most of the analytical methods published in the literature are to a certain extent in-house validated
(Delgrado et al., 1996; da Motta and Soares, 2000a,b; Diana di Mavungu et al., 2009; Asam et al., 2009,
2010). No information on interlaboratory validation, standardisation of the analytical methods or
conduction of proficiency tests is available.
In summary, several chromatography based techniques are suitable for Alternaria toxin quantification in
foods and feeds. LC coupled to (tandem) MS has become the method of choice due to its sensitivity,
selectivity and specificity. Limiting factors, however, are sometimes the poor efficiency of sample
cleanup, the availability of (sufficient) amounts of standards and the lack of reference materials.
3.
Occurrence of Alternaria toxins in food and feed
3.1.
Previously reported occurrence results of Alternaria toxins in food
The natural occurrence of Alternaria toxins in food has been previously reviewed by Bottalico and
Logrieco (1998), Scott (2001), Ostry (2008) and Fernandez-Cruz et al. (2010). According to Ostry
(2008), the maximum levels of Alternaria toxins were reported for marketed products (concentration
range 1 to 1000 g/kg). The higher levels were found in food samples visibly infected by Alternaria rot
and thus these foods were not suitable for human consumption. An overview of the occurrence of AOH,
AME, ALT, ATX-I, TeA and TEN in foodstuffs is given in Appendix B Table B1 and a summary of the
occurrence data reported in Europe is given in Table 3. Due to the scarce number of European studies
reporting Alternaria toxin concentrations in food samples, this chapter also describes studies carried out
in the countries outside Europe. Only data for which the LOD or LOQ was available are reported. The
typical LODs and LOQs for Alternaria toxins in food are described in Section 2.2.
In Europe, the analyses of approximately 850 food samples for Alternaria toxins, mainly for AOH and
AME and to a lesser extent for ALT, ATX-I, TeA and TEN, have been reported in the literature
between 1983 and 2010.
AOH was quantified in 13 % of all the food samples reported. The AOH concentrations ranged from
< 0.01 to 290 g/kg with the highest level being found in lentils (Ostry et al., 2004). The second-highest
levels were found in oilseeds (104 g/kg) (Krlov et al., 2006) and in a tomato paste sample (25 g/kg)
(Asam et al., 2010). The other studies reported AOH levels well below 10 g/kg in fruit juices, wines,
must, cereals, vegetables (tomato products, carrots and other vegetables) and in edible oils (Wittkowski
EFSA Journal 2011;9(10):2407
18
et al., 1983; Visconti et al., 1986; Gyrin and Szteke, 1995; Delgado et al., 1996; Delgado and GomezCorvods, 1998; Solfrizzo et al., 2004; Ostry et al., 2007; Asam et al., 2009, 2010; Ackermann et al.,
2011). Asam et al. (2009) reported that in general fruit juices showed low concentrations of AOH and
AME, but higher values of both toxins were found in wine and vegetable juices.
AME was quantified in 6 % of all the food samples reported in literature and the concentrations ranged
from < 0.01 to 30 g/kg. The highest concentration (30 g/kg) was reported for oilseeds (linseed and
fibre flax seed) (Krlov et al., 2006) followed by tomato paste (maximum 5.3 g/kg) (Asam et al.,
2010). The other studies reported AME levels as being well below 2 g/kg in fruit juices, wines, must,
cereals and vegetables (tomato products, carrots and lentils) (Wittkowski et al., 1983; Visconti et al.,
1986; Gyrin and Szteke, 1995; Delgado et al., 1996; Delgado and Gomez-Corvods, 1998; Solfrizzo et
al., 2004; Ostry et al., 2004, 2007; Asam et al., 2009, 2010).
Only 0.6 % of the samples reported in the literature were quantified for ALT. The concentrations varied
between < 1 and 9 g ALT/kg. The highest concentrations were found in oilseeds (Krlov et al., 2006).
No other quantifiable concentrations for ALT in wine, must, grape juice, edible oils, lentils and
vegetables were reported (Wittkowski et al., 1983; Visconti et al., 1986; Solfrizzo et al., 2004; Ostry et
al., 2004, 2007).
ATX-I was not found in any of the food samples analysed (Wittkowski et al., 1983; Visconti et al.,
1986; Solfrizzo et al., 2004; Ostry et al., 2004, 2007).
TeA was quantified in 5 % of the food samples analysed and the concentrations ranged from < 2 to 851
g/kg. The highest concentration was reported in cereals (maximum 851 g/kg, mean 49 g/kg)
followed by bottled beers (maximum of 175 g/kg, mean 11 g/kg (Siegel et al., 2009, 2010b). The
average concentration in beer type varied between 5.6 g/kg in wheat beer (n = 8) and 9.2 g/kg in bock
beer (n = 6) when the maximum level was omitted as an outlier. A concentration of 25 g/kg of TeA
was also found in a sample of cornflakes (Aresta et al., 2003). No TeA levels was detected in wine,
must, grape juice, organic carrots, lentils, olive husks and olive oils (Visconti et al., 1986; Solfrizzo et
al., 2004; Ostry et al., 2004, 2007).
In North America, AOH, AME and TeA were analysed in 120 food samples of fruit juices and wines
(Scott et al., 1997, 2006; Lau et al., 2003; Abramson et al., 2007). Regarding the studies of Scott et al.
(2006) and Lau et al. (2003), only the results obtained by LC-MS or LC-MS/MS are reported in
Appendix B Table B1.
AOH was quantified in 46 % of the samples, concentrations ranging from < 0.01 to 7.41 g/L. The
highest concentration was found in a sample of imported red wine (Scott et al., 2006), followed by other
fruit beverages (5.6 g/L) (Lau et al., 2003), apples juice and red wine (5 g/L) (Scott et al., 1997,
2006). The other AOH levels found in these three studies were 3 g/L.
AME was quantified in 38 % of the samples (n = 120) with the concentration range of < 0.01- 39.5 g
AME/L. Scott et al., (2006) reported the highest concentration in a sample of red grape juice
(39.5 g/L). The other AME levels reported were 2 g/L in fruit juice and wine samples (Scott et al.,
1997, 2006; Lau et al., 2003).
TeA was not quantified in the 26 ice-wine samples analysed by Abramson et al. (2007).
In South America, AOH, AME and TeA were analysed in 160 food samples of tomato and tomato
products (Appendix B Table B1) (da Motta and Valente Soares, 2001; Terminiello et al., 2006).
AOH was quantified in 3 % of the samples of tomato and tomato products, concentrations ranging from
< 5 to 8756 g/kg. The highest concentration was found in a tomato puree sample (Terminiello et al.,
EFSA Journal 2011;9(10):2407
19
2006). In the study of da Motta and Valente Soares (2001), the AOH levels were 5 g/kg in tomato
and tomato products.
AME was quantified in 13 % of the samples (n = 160) with the concentration range of < 2-1734 g
AME/kg. Terminiello et al. (2006) reported the highest concentration in a tomato puree sample
(1734 g AME/kg) while da Motta and Valente Soares (2001) did not quantify AME in any of the
142 tomato and tomato products samples analysed.
TeA was quantified in 21 % of the food samples analysed. The concentrations varied between < 11 and
4021 g/kg. Again the highest concentrations were reported for tomato puree samples (7 samples were
1000 g/kg) (Terminiello et al., 2006). TeA was also found in tomato pulp samples (39-111 g
TeA/kg) but the other tomato products were negative for TeA (da Motta and Valente Soares, 2001).
No available data were identified from Africa, Asia, Australia.
The CONTAM Panel also noted that several studies reported results on mouldy foods which were
severely contaminated with Alternaria toxins (Stinson et al., 1981; Wittkowski et al., 1983; Stack et al.,
1985; Visconti et al., 1986; Logrieco et al., 1988; Gyrin and Szteke, 1995; Sulyok et al., 2010). The
highest concentrations were up to 2870 g/kg in an olive sample for AME (Visconti et al., 1986),
58800 g/kg in an apple sample for AOH (Stinson et al., 1981), 1100 g/kg in a tomato sample for ALT
(Stinson et al., 1981) and 174000 g/kg in a mandarin sample for TeA (Logrieco et al., 1988). Since
mouldy foods are not appropriate for human consumption these studies were not further considered. The
occurrence data on food reported in Europe are summarised in Table 3.
Summary of the occurrence of Alternaria toxins in food reported from Europe.
Table 3:
Alternaria
toxin
N of
total
samples
N of
positive
samples
Positive
samples
(%)
Maximum
concentration
(g/kg)
Food type reported

for the maximum
concentration
AOH
859
109
13
290
Lentils
AME
750
43
30
ALT
313
0.6
ATX-I
331
n.d.
Oilseeds
(linseed
and fibre flax seed)
Oilseeds
(linseed
and fibre flax seed)
-
TeA
397
19
851
Cereals
Reference
(reporting the
maximum
concentration)
Ostry et al.
(2004)
Krlov et al.
(2006)
Krlov et al.
(2006)
Siegel et
(2009)
al.
AOH: alternariol; AME: alternariol monomethyl ether; ALT: altenuene ATX-I: altertoxins I; TeA: tenuazonic acid; N: number
of samples; n.d.: not detected; -: not available.
3.2.
Currently reported occurrence results of Alternaria toxins in food
3.2.1.
Data collection summary
The Dietary and Chemical Monitoring Unit (DCM) (former Data Collection and Exposure Unit,
DATEX) call for data on Alternaria toxins in food and feed6 was launched in October 2010. European
national food authorities and similar bodies, research institutions, academia, food and feed business
operators and any other stakeholders were invited to submit analytical data on Alternaria toxins in food
http://www.efsa.europa.eu/en/dataclosed/call/datex101020b.htm
EFSA Journal 2011;9(10):2407
20
and feed by January 2011. The data submission to EFSA followed the requirements of the EFSA
Guidance on Standard Sample Description for Food and Feed (EFSA, 2010a).
In total, 10,330 data on Alternaria toxins in food and agricultural commodities were received from
national food authorities or research institutions in the Czech Republic (n = 540) and Germany
(n = 9790). Data reported were on samples collected from 2005 to 2010.
In addition to the data collected from the Member States, occurrence data on Alternaria toxins in food
were collected from the literature and included in the database. For this purpose, 1,832 occurrence data
on individual samples were extracted from the following publications: Gyrin and Szteke (1995);
Delgado et al. (1996); Delgado and Gomez-Corvods (1998); Aresta et al. (2003); Solfrizzo et al.
(2004); Krlov et al. (2006), Asam et al. (2009); Ostry et al. (2007); Siegel et al. (2009); Asam et al.
(2010); Siegel et al. (2010b). The Member States which provided data to EFSA within the call for data
confirmed that data from the aforementioned publications are not included in their database thus there
were no duplicate data. Overall, the database on Alternaria toxins included 12,162 observations for
AOH (n = 2355), AME (n = 2279), ALT (n = 1811), ATX-I (n = 1279), TeA (n = 1951), TEN
(n = 1388), AAL- toxins (n = 863) and Alternaria toxins unspecified (n = 236). The distribution of food
samples over the sampling countries and sampling years is presented in Table 4.
To ensure the quality of data included in the assessment, several data cleaning and validation steps were
applied. Analytical results with incomplete or incorrect description of the relevant variables (e.g.
parameter type, food classification, LOD or LOQ) were not included in the data sets used in this
assessment. In total, 432 observations were excluded from the assessment as follows: 236 observations
on Alternaria toxins unspecified, 192 observations on grains of unknown end-use and 4 samples
analysed for TeA by a method with LOQ = 154 g/kg (all left-censored data). The final data set
considered in the assessment included 11,730 observations on AOH (n = 2291), AME (n = 2215), ALT
(n = 1747), ATX-I (n = 1279), TeA (n = 1947), TEN (n = 1388) and sum of AAL-toxins (n = 863) in
food samples.
Table 4:
years.
Distribution of food samples for Alternaria toxins over the sampling countries and sampling
Czech Republic
AOH
AME
ALT
ATX-I
TeA
TEN
AALtoxins
Germany
Italy Poland
2002 2004 2010 2005 2006 2007 2008 2009 2010 2003
96
264 521 477
84
39
116 191 155
96
275 402 477
84
39
116 191 155
96
253 372 477
84
39
116 155 155
212 369 432
191 155
96
321 402 476
1
39
154 152
96
244 265 477
226 260 377
1995
75
75
Spain
European
Union(a)
1993 1995
2002
7
266
32
7
266
266
266
AOH: alternariol; AME: alternariol monomethyl ether; ALT: altenuene ATX-I: altertoxins I; TeA: tenuazonic acid; TEN:
tentoxin; AAL-toxins: Alternaria alternata f. sp. lycopersici toxins; (a): For some data from the literature there were more
sampling countries given for the pool data. Therefore it was not possible to identify the sampling country for each sample.
3.2.2.
Distribution of samples across food groups
The food samples were classified according to the FoodEx classification system (EFSA, 2011a).
FoodEx is a food classification system developed by the DCM Unit in 2009 with the objective of
simplifying the linkage between occurrence and food consumption data when assessing the exposure to
hazardous substances. It contains 20 main food groups (first level), which are further divided into
subgroups having 140 items at the second level, 1261 items at the third level and reaching about
1800 end-points (food names or generic food names) at the fourth level. The spread of the analytical
EFSA Journal 2011;9(10):2407
21
results for Alternaria toxins across the several FoodEx groups prevented calculation of summary
statistics at a detailed level of the food classification system.
The distribution of samples across food groups is shown in Table 5. The highest number of data
available for each Alternaria toxin were in the food groups Grain and grain-based products,
Vegetables and vegetable products, Legumes, nuts and oilseeds, Fruit and vegetable juices,
Vegetable oils, Fruit and fruit products. The other food groups were less represented. In some food
groups, data were available only on a limited number of food commodities and not all food items in the
group were represented. More detailed distribution of food samples across food groups is presented in
Section 3.2.4.
Table 5:
Distribution of food samples for Alternaria toxins across the food groups.
Food group
Grains and grain-based products
Grains for human consumption
Grain milling products
Bread and rolls
Breakfast cereals
Fine bakery wares
Pasta (raw)
Vegetables and vegetable products
Starchy roots and tubers
Legumes, nuts and oilseeds
Fruit and fruit products
Sugar and confectionary
Vegetable oils
Fruit and vegetable juices
Non-alcoholic beverages
Alcoholic beverages
Herbs, spices and condiments
Food for infants and small children
Products for special nutritional use
Composite food
Snacks, desserts, and other foods
Total
AOH
AME
ALT
ATX-I
TeA
TEN
911
265
165
276
99
84
21
392
37
254
106
5
100
299
15
54
33
49
21
3
12
911
265
165
276
99
84
21
382
37
254
86
5
100
276
15
49
15
49
21
3
12
899
265
156
276
96
84
21
108
1
254
86
5
100
172
3
33
13
49
21
3
0
641
185
141
160
52
82
20
341
0
147
67
5
17
8
0
0
0
29
21
3
0
810
265
172
169
98
84
21
364
37
168
86
5
100
194
3
76
31
49
21
3
0
676
264
114
130
69
77
21
108
0
170
86
5
99
158
3
7
13
49
12
2
0
AALtoxins
559
184
106
129
42
77
20
55
0
125
49
5
0
8
0
0
0
48
12
2
0
2291
2215
1747
1279
1947
1388
863
AOH: alternariol; AME: alternariol monomethyl ether; ALT: altenuene ATX-I: altertoxins I; TeA: tenuazonic acid; TEN:
tentoxin; AAL-toxins: Alternaria alternata f. sp. lycopersici toxins
3.2.3.
Analytical methods used
Data on Alternaria toxins in food were obtained by LC-MS/MS (78 %), HPLC-UV (9.5 %), HPTLC
(2.6 %), HPLC-FL (2.1 %) and ELISA (1.4 %). For the remaining 6.4 % of the food samples, the
analytical method was not reported.
EFSA Journal 2011;9(10):2407
22
LODs and LOQs were reported for 97 % and 89 % of observations, respectively. The LODs and LOQs
varied with the toxin, the method applied, the food matrix and the laboratory. An overview on the
ranges of LODs and LOQs reported for the Alternaria toxins is presented in Table 6.
The reported data on Alternaria toxins in food were characterised by a high proportion of left-censored
data (results below LOD/LOQ) ranging from 87 % up to 100 % for the different compounds.
Table 6: Summary statistics on the limits of detection and limits of quantification reported for
Alternaria toxins.
Toxin
Limit of detection (g/kg)
ALT
AOH
AME
ATX I
AAL-toxins
TEN
TeA
Limit of quantification (g/kg)
Min
P25
Median
P75
Max
Min
P25
Median
P75
Max
1708
2133
2144
1279
863
1388
1868
1.0
0.01
0.01
10
15
2.0
2.0
3.0
5.0
1.0
15
15
3.0
20
8.0
6.0
1.0
15
15
4.0
25
8.0
6.0
3(a)
15
15
4.0
25
15
6.0
6.0
20
15
4.0
25
1747
1883
1867
1013
863
1388
1676
1.5
0.03
0.03
20
30
4.0
7.5
6.0
8.0
2.0
30
30
6.0
30
16
12
2.0
30
30
8.0
50
16
12
2(a)
30
30
8.0
50
30
12
15
30
30
8.0
50
ALT: altenuene; AOH: alternariol; AME: alternariol monomethyl ether; ATX-I: altertoxins I; AAL-toxins: Alternaria
alternata f. sp. lycopersici toxins; TEN: tentoxin; TeA: tenuazonic acid; N: number of samples; Min: minimum; P25: 25th
percentile; P75: 95th percentile; Max: maximum; (a): The P75 value of the limit of detection is higher than the P75 value of the
limit of quantification because the calculation were performed on slightly different datasets.
3.2.4.
Occurrence data by food category
In the analysis of Alternaria toxin occurrence data the left-censored data were treated by the substitution
method as recommended in the Principles and Methods for the Risk Assessment of Chemicals in Food
(WHO, 2009). The same method is indicated in the EFSA scientific report Management of leftcensored data in dietary exposure assessment of chemical substances (EFSA, 2010b) as an option in
the treatment of left-censored data. The guidance suggests that the lower-bound (LB) and upper-bound
(UB) approach should be used for chemicals likely to be present in the food (e.g. naturally occurring
contaminants, nutrients and mycotoxins). The LB is obtained by assigning a value of zero (minimum
possible value) to all samples reported as lower than the LOD (< LOD) or LOQ (< LOQ). The UB is
obtained by assigning the numerical value of LOD to values reported as < LOD and LOQ to values
reported as < LOQ (maximum possible value), depending on whether LOD or LOQ is reported by the
laboratory.
The analytical results were transmitted by the data providers as either corrected or not corrected for
recovery. Where results were not corrected by the data provider a correction has been applied by using
the reported recovery rate. Where recovery was not available, no correction has been applied.
An overview on the number of samples and toxin concentration for AOH, AME, TeA and TEN in food
groups (FoodEx level 1), is given in Tables 7 to 10 . Mean concentrations in LB and UB for more
detailed food groups are given in Table 11. However, since the number of samples available in each
food-subgroup was limited, the mean concentration values should be interpreted with caution.
The vast majority of data received for all toxins were on grains and grain-based products. This food
group covers grains for human consumption, grain milling products, bread and rolls, pasta, breakfast
cereals and fine bakery wares. Within the group, the highest mean concentrations were observed in
grains as follows: AOH - spelt, oats, rice; AME - oats, rice; TeA - wheat, barley, rye, spelt, oats and
EFSA Journal 2011;9(10):2407
23
rice; TEN - rye. In grain milling products the mean concentrations were generally lower compared to
grains. In the food group Bread and rolls, only 2 samples (unleavened bread) were positive for AME
(n = 276) and TeA (n = 169), respectively. In the food groups Pasta, Breakfast cereals and Fine
bakery wares (Biscuits) AOH, AME and TeA were found in maximum one or two samples.
In the food group Vegetables and vegetable products AOH, AME and TeA were found in tomato
products (tomato puree, tomato ketchup), tea and herbs for infusions and sesame paste.
In the food group Legumes, nuts and oilseeds the sunflower seeds had the highest mean concentrations
of AOH, AME, TeA, TEN followed by sesame seeds for AOH, AME and TeA.
In Fruit and fruit products, the number of samples analysed for the individual foods was very limited
thus difficult to draw a firm conclusion. TeA had the highest contamination frequency and mean
concentrations in figs (LB mean = 604 g/kg; UB mean = 610 g/kg).
Among vegetable oils, AOH, AME, TeA and TEN were found in sunflower oil, thistle oil and, with the
exception of TEN, in sesame oil.
The food group Sugar and confectionary included only 5 samples. However, AME and TeA were
found in relatively high concentrations in one sample of Halva (a sunflower seed dessert). For
simplification, in the table used for exposure calculation (Table 11) Halva was moved into the group
Vegetables and vegetable products.
Mean concentrations of AOH and AME in fruit and vegetable juices ranged from 0.01 g/kg (LB
mean) to 3 g/kg (UB mean). TeA was present in higher concentrations in vegetable juices and in
particular in tomato juice (LB mean = 26 g/kg; UB mean = 69 g/kg). TEN was not found in any of
the fruit and vegetable juices analysed.
In the group Alcoholic beverages, AOH and AME were reported in wine whereas TeA was reported in
beer. To note that only one beer sample was analysed for AOH and AME thus no conclusion can be
drawn on the presence of these Alternaria toxins in beer. Furthermore, no data were available on the
presence of TEN in wine or beer.
The food group Herbs, spices and condiments included mainly samples of tomato ketchup. For
simplification, in the table used for exposure calculation tomato ketchup was assigned to the food group
Vegetables and vegetable products and is further discussed with this group.
In the food group Food for infants and small children, two samples contained TeA whereas AOH,
AME and TEN were not found in any of the samples analysed. Products for special nutritional use
covered samples on Formulas for metabolic disorders. No further information was available to better
characterise the samples. AME and TeA were reported in one sample.
Due to the absence of results above the LOD or LOQ, no statistics on the ALT, ATX I and sum of AAL
toxins concentration is provided. ATX I was found in only two samples: one sesame seed (80 g/kg)
and one sesame paste (41 g/kg). ALT and AAL toxins were not detected in any of the samples
analysed.
EFSA Journal 2011;9(10):2407
24
Table 7:
Concentrations of AOH across food groups (FoodEx level 1).
Food group
N(a)
LC
Concentration range (LB UB)(b) (g/kg or g/L)

Mean
P50
P75
P95
Maximum
911
97%
1.8 - 7.3
0.0 - 6.0
0.0 - 6.0
0.0 - 6.0
256
392
92%
1.1 - 5.8
0.0 - 5.0
0.0 - 5.0
6.2 - 12
100
(c)
(c)
(c)
5.0(d)
1200
37
100%
0.0 - 5.0
254
87%
22 - 26
0.0 - 6.0
0.0 - 6.0
70 - 70
106
89%
2.7 - 7.3
0.0 - 6.0
0.0 - 6.0
2.0 - 12
151
100%
0.0 - 7.2
-(c)
-(c)
-(c)
12(d)
Vegetable oils
100
93%
0.9 - 5.6
0.0 - 5.0
0.0 - 5.0
11 - 11
18
299
87%
0.4 - 2.7
0.0 - 3.0
0.0 - 3.0
2.4 - 6.0
13
(c)
(c)
(c)
3.0(d)
15
100%
0.0 - 3.0
Alcoholic beverages
54
83%
0.4 - 1.7
0.0 - 1.5
0.0 - 1.5
2.7 - 3.0
4.9

Composite food
33
49
21
3
12
42%
100%
100%
100%
100%
1.5 - 2.7
0.0 - 6.0
0.0 - 6.3
0.0 - 6.0
0.0 - 3.0
1.6 - 3.0
-(c)
-(c)
-(c)
-(c)
2.5 - 3.0
-(c)
-(c)
-(c)
-(c)
4.5 - 5.0
-(c)
-(c)
-(c)
-(c)
5.0
6.0(d)
12(d)
6.0(d)
3.0(d)
AOH: alternariol; N: number of samples; LB: lower bound; UB: upper bound; (a): If N < 60 then the calculated P95 should be
considered as an indicative value only due to the limited number of data (EFSA, 2011b); LC: left censored data (values below
the limit of detection or limit of quantification); (b): Range indicates lower-bound and upper-bound values. P50:
50th percentile; P75: 75th percentile; P95: 95th percentile; (c): not calculated where all data were left-censored or the number of
data was very limited; (d): value represent the left-censoring limit.
Table 8:
Concentrations of AME across food groups (FoodEx level 1).
Food group
N(a)
LC

911
382
98%
92%
0.37 - 1.99
1.4 - 8.65
0.0 - 1.0
0.0 - 10
0.0 - 1.0
0.0 - 10
0.0 - 6.0
3.8 - 10
86
280
37
100%
0.0 - 1.0
-(c)
-(c)
-(c)
1.0(d)
254
73%
11 - 12
0.0 - 2.0
3.0 - 3.0
38 - 38
440
86
93%
1.4 - 2.4
0.0 - 1.0
0.0 - 1.0
8.4 - 8.4
42
1.3 - 2.3
(c)
(c)
(c)
6.7

Mean
P50
P75
P95
Maximum
80%
Vegetable oils
100
84%
3.4 - 6.1
0.0 - 3.0
0.0 - 5.0
25 - 25
85
276
97%
0.02 - 1.3
0.0 - 1.0
0.0 - 1.0
0.0 - 3.0
3.0
15
100%
0.0 - 2.6
-(c)
-(c)
-(c)
3.0(d)
Alcoholic beverages
49
90%
0.02 - 0.9
0.0 - 1.5
0.0 - 1.5
0.2 - 1.5
1.5(d)
15
93%
0.03 - 0.9
-(c)
-(c)
-(c)
1.0(d)
49
100%
0.0 - 1.0
-(c)
-(c)
-(c)
1.0(d)
21
95%
0.16 - 1.1
0.0 - 1.0
0.0 - 1.0
0.0 - 1.0
3.3
Composite food
3
12
100%
100%
0.0 - 1.0
0.0 - 3.0
-(c)
-(c)
-(c)
-(c)
-(c)
-(c)
1.0(d)
3.0(d)
AME: alternariol monomethyl ether; N: number of samples; LB: lower bound; UB: upper bound; (a): If N < 60 then the
calculated P95 should be considered as an indicative value only due to the limited number of data (EFSA, 2011b); LC: left
censored data (values below the limit of detection or limit of quantification); (b): Range indicates lower-bound and upperbound values. P50: 50th percentile; P75: 75th percentile; P95: 95th percentile; (c): not calculated where all data were leftcensored or the number of data was very limited; (d): value represent the left-censoring limit.
EFSA Journal 2011;9(10):2407
25
Table 9:
Concentrations of TeA across food groups (FoodEx level 1).
Food group
N(a)
LC

810
364
94%
88%
6.7 - 32
15 - 34
0.0 - 25
0.0 - 20
0.0 - 25
0.0 - 25
48 - 50
90 - 90
851
520
37
100%
0.0 - 10
-(c)
-(c)
-(c)
10(d)
168
55%
333 - 349
0.0 - 50
86
76%
158 - 176
0.0 - 25
0.0 - 50
378 - 378
8700
(c)
(c)
(c)
340

Mean
P50
P75
P95
Maximum
220 - 220 2000 - 2000

-
80%
68 - 88
Vegetable oils
100
64%
35 - 42
0.0 - 15
33 - 33
227 - 227
390
194
98%
2.4 - 14
0.0 - 5.0
0.0 - 20
0.0 - 50
286
(c)
(c)
(c)
5.0(d)
0.0 - 8.0
19 - 19
175
100%
0.0 - 5.0
Alcoholic beverages
76
80%
4.7 - 10
5400
0.0 - 7.8
31
65%
30 - 54
0.0 - 40
59 - 59
116 - 116
163
49
96%
3.7 - 30
0.0 - 25
0.0 - 25
0.0 - 50
110

Composite food
21
3
95%
100%
20 - 45
0.0 - 25
0.0 - 25
-(c)
0.0 - 25
-(c)
0.0 - 50
-(c)
424
25(d)
TeA: tenuazonic acid; N: number of samples; LB: lower bound; UB: upper bound; (a): If N < 60 then the calculated P95
should be considered as an indicative value only due to the limited number of data (EFSA, 2011b); LC: left censored data
(values below the limit of detection or limit of quantification); (b): Range indicates lower-bound and upper-bound values. P50:
Table 10: Concentrations of TEN across food groups (FoodEx level 1).
Food group
N(a)
LC

676
108
97%
99%
170
86
Vegetable oils
99
158

Mean
P50
P75
P95
Maximum
0.29 - 4.1
0.09 - 3.9
0.0 - 4.0
0.0 - 4.0
0.0 - 4.0
0.0 - 4.0
0.0 - 8.0
0.0 - 4.0
74%
47 - 50
0.0 - 4.0
5.0 - 8.0
330 - 330
880
100%
0.0 - 3.7
-(c)
-(c)
-(c)
4.0(d)
100%
0.0 - 4.8
-(c)
-(c)
-(c)
8.0(d)
80%
3.4 - 5.1
0.0 - 2.0
0.0 - 4.0
26 - 26
67
0.0 - 2.9
(c)
(c)
(c)
4.0(d)
(c)
(c)
(c)
3.0(d)
100%
38
9.2
100%
0.0 - 3.0
Alcoholic beverages
100%
0.0 - 3.0
-(c)
-(c)
-(c)
3.0(d)
13
100%
0.0 - 3.0
-(c)
-(c)
-(c)
3.0(d)
49
100%
0.0 - 4.0
-(c)
-(c)
-(c)
4.0(d)
0.0 - 4.0
0.0 - 4.0
(c)
(c)
(c)
4.0(d)
4.0(d)

Composite food
12
2
100%
100%
-(c)
-(c)
-(c)
TEN: tentoxin N: number of samples; LB: lower bound; UB: upper bound; (a): If N < 60 then the calculated P95 should be
considered as an indicative value only due to the limited number of data (EFSA, 2011b); LC: left censored data (values below
the limit of detection or limit of quantification); (b): Range indicates lower-bound and upper-bound values. P50:
EFSA Journal 2011;9(10):2407
26
Table 11: Mean concentration (g/kg or g/L) of AOH, AME, TeA and TEN in the food groups
considered for the calculation of dietary exposure. If all data in a food group were left censored, or the
number of samples was below 5, the food group was not included in the calculation of dietary exposure
(grey shaded cells).
Food group
AOH
AME
TeA
TEN
N LC Mean Mean N LC Mean Mean N LC Mean Mean N LC Mean Mean
LB UB
LB UB
LB
UB
LB
UB
Grains for human
265 242
consumption (unspecified)
Wheat grain
67 64
Barley grain
36 32
Rye grain
31 31
Spelt grain
13 10
Oats, grain
41 30
Rice
44 42
Grain milling products
165 164
(unspecified)
Wheat milling products
54 54
Rye milling products
26 26
Buckwheat milling
1
1
products
Spelt milling products
11 10
Unleavened bread, crisp
7
7
bread and rusk
Pasta (Raw)
21 20
Breakfast cereals
99 97
(unspecified)
Cereal flakes
93 91
Biscuits (cookies)
44 44
Tomatoes
10 10
Tea and herbs for infusions
23 17
(solid)
Vegetable products
83 59
(unspecified)
Tomato pure
56 33
Tomato ketchup
32 13
Sun-dried tomatoes
6
6
Sesame paste (Tahini)
15 15
Halva
2
2
Hops (dried)
1
1
Oilseeds
Oilseeds (unspecified)
139 105
Linseed
3
3
Sesame seed
45 35
Sunflower seed
74 54
Pumpkin seeds
4
4
EFSA Journal 2011;9(10):2407
6.1
11
265 248 1.3
2.2
265 233 13
37
264 248 0.71
4.2
0.62
1.1
0.0
25
23
6.1
5.7
5.5
4.8
29
26
12
67
36
31
13
41
44
1.0
1.0
1.0
1.1
5.1
3.9
67
36
31
13
41
44
3.0
7.3
24
11
26
16
26
30
49
33
50
43
67
36
31
13
41
44
0.08
0.0
4.5
0.0
1.0
0.0
3.3
2.9
7.4
3.7
4.4
4.2
0.02
5.7
165 165 0.0
1.0
172 165 8.1
33
114 113 0.07
4.3
0.0
0.0
5.7
5.6
54
26
54
26
0.0
0.0
1.0
1.1
59
26
58
22
1.4
16
26
45
28
23
28
22
0.0
0.37
4.1
5.4
0.0
5.0
0.0
1.0
301
318
0.0
2.0
0.37
5.8
11
11
0.0
0.9
12
12
0.0
26
0.0
4.0
0.0
6.0
0.81 1.7
16
14
8.1
39
0.0
4.6
0.62
6.3
21
21
0.0
21
20
2.4
34
21
21
0.0
3.9
0.29
6.0
99
98
0.01 0.98
98
96
2.5
28
69
69
0.0
4.1
0.31
0.0
6.0
6.0
93
44
92
44
0.01 0.98
0.0 1.0
92
44
90
43
2.7
1.3
28
27
63
37
63
37
0.0
0.0
4.1
4.0
0.0
5.7
10
10
0.0
1.0
10
34
54
10
10
0.0
3.9
8.2
13
23
13
15
16
13
26
45
23
23
0.0
4.4
2.7
6.5
73
54
2.4
3.6
65
30
73
86
65
64
0.14
3.7
3.6
1.5
0.0
0.0
0.0
0.0
6.5
2.8
6.0
6.4
9.0
6.0
46
14
6
15
2
1
39
13
6
4
1
1
0.56
0.03
0.0
9.8
3.4
0.0
1.9
0.96
1.0
10
4.4
1.0
43
31
6
15
2
1
10
20
6
14
1
0
106
30
0.0
9.3
170
68
113
54
29
33
183
68
43
13
6
15
2
1
43
13
5
15
2
1
0.0
0.0
1.5
0.0
0.0
0.0
3.3
3.0
5.5
4.0
6.0
4.0
40
0.0
7.0
66
0.0
45
6.0
12
71
6.0
139
3
45
74
4
71
3
11
46
4
20
0.0
21
23
0.0
20
1.3
21
24
1.0
137
3
45
72
4
61
2
30
14
3
408
18
60
731
22
422
35
80
738
41
139
3
45
74
4
95
2
45
32
4
58
4.2
0.0
106
0.0
60
6.9
3.8
108
4.0
66
36
31
13
32
40
0.03
0.0
0.0
0.0
4.3
3.0
1.0
65
33
21
11
35
38
66
36
18
13
39
44
27
Table 11: Continued.

Food group
AOH
AME
TeA
TEN
N LC Mean Mean N LC Mean Mean N LC Mean Mean N LC Mean Mean
LB UB
LB UB
LB
UB
LB
UB

106 94
(unspecified)
Pears
12 9
Apricots
4
4
Berries and small fruits
9
8
Figs
22 21
Dried vine fruits (currants,
14 14
raisins and sultanas)
Jam
20 19
Other fruit products
(excluding beverages)
17 11
(unspecified)
Fruit, pure
10 4
Vegetable oils
Vegetable oil (unspecified) 99 92
Peanut oil
6
6
Pumpkinseed oil
1
1
Rape seed oil
30 30
Sesame oil
12 9
Sunflower oil
18 16
Thistle oil
15 14
299 261
(unspecified)
Fruit juice (unspecified)
242 219
Apple juice
127 112
Orange juice
25 20
Grapefruit juice
9
8
Grape juice
52 50
Vegetable juice
37 22
(unspecified)
Tomato juice
25 11
Alcoholic beverages
Beer and beer-like
1
1
beverage
Wine
32 24
Food for infants and small
49 49
children (unspecified)
Cereal-based food for
infants and young children 37 37
(unspecified)
Formulas for metabolic
21 21
disorders
2.7
7.3
86
80
1.4
2.4
86
65
158
176
86
86
0.0
3.7
5.4
0.0
6.8
6.9
10
6.0
12
13
12
4
9
22
9
4
8
21
5.5
0
4.6
0.18
6.3
1.0
5.6
1.2
12
4
9
22
12
3
8
3
0.0
18
25
604
25
43
47
610
12
4
9
22
12
4
9
22
0.0
0.0
0.0
0.0
4.0
4.0
4.0
4.0
0.0
6.4
14
13
0.6
1.7
14
14
0.0
30
14
14
0.0
4.0
0.05
3.0
10
10
1.0
10
10
0.0
15
10
10
0.0
2.0
0.53
2.0
1.0
0.0
5.0
0.0
3.0
0.9
1.3
0.93
0.0
0.0
0.0
2.9
1.6
1.0
5.6
5.0
5.0
5.0
6.9
6.2
5.7
99
6
1
30
12
18
15
83
6
1
30
3
14
13
3.4
0
0
0
20
3.1
2.0
6.1
3.0
3.0
3.0
21
5.7
4.8
99
6
1
30
12
18
15
63
5
1
27
6
3
4
35
4.0
0.0
3.3
11
113
81
43
14
10
15
17
115
84
98
6
1
30
12
18
15
78
6
1
30
12
3
10
3.5
0.0
0.0
0.0
0.0
16
3.5
5.2
2.0
2.0
2.0
2.0
16
5.4
0.43
2.7
276 268 0.02 1.3
194 190 2.4
14
158 158 0.0
2.9
0.38
0.21
1.3
1.1
0.44
2.6
2.3
2.9
2.9
3.0
235
115
25
9
57
231
113
23
9
57
0.01
0.02
0.02
0.0
0.0
168
80
23
9
51
166
79
23
9
50
0.5
0.7
0.0
0.0
0.41
9.2
7.2
20
20
6.3
147
73
22
9
38
147
73
22
9
38
0.0
0.0
0.0
0.0
0.0
2.8
3.0
2.0
2.0
3.0
0.98
2.9
21
17
0.11 1.4
23
21
17
54
0.0
4.0
1.1
2.1
0.17 2.2
15
13
26
69
0.0
0.5
0.0
0.10
43
28
8.2
13
0.66
1.6
27
23
0.03 0.76
13
13
0.0
7.5
0.0
6.0
49
49
0.0
1.0
49
47
3.7
30
49
49
0.0
4.0
0.0
6.0
37
37
0.0
1.0
37
36
1.9
29
37
37
0.0
4.0
0.0
6.3
21
20
0.16 1.1
21
20
20
45
12
12
0.0
4.0
1.2
0.91
1.1
1.0
1.0
AOH: alternariol; AME: alternariol monomethyl ether; TeA: tenuazonic acid; TEN: tentoxin; N: number of samples; LC: left
censored data (values below the limit of detection or limit of quantification); LB: lower bound; UB: upper bound; - : no data
available.
To evaluate the co-occurrence of Alternaria toxins, the subset of samples where two toxins co-occurred
were extracted from the database and the ratio between the concentrations observed for the individual
EFSA Journal 2011;9(10):2407
28
toxins were calculated. Due to the limited number of samples where two Alternaria toxins co-occurred
across all food groups (less than 70 samples) it was difficult to draw a firm conclusion. For example, the
ratio between AOH and AME (n = 67) ranged from 0.1 to 38; between AOH and TeA (n = 63) ranged
from 0.3 to 122.
3.3.
Previously reported occurrence results of Alternaria toxins in feed
The published occurrence data in feed and agricultural commodities from Europe and other regions in
the world are reported in Appendix B, Table B2. Only data for which the LOD or LOQ was available
are reported. The typical LODs and LOQs for Alternaria toxins in feed are described in Section 2.2.
Samples showing visible fungal growth of Alternaria species or which were examined to be infected by
Alternaria species were also reported in Appendix B, Table B2, because these may be used as feed.
These infections may result from wet weather conditions during harvest and wetting of the seeds leading
to higher infection intensity, and therefore these seeds are also called weathered or weather-damaged. A
summary of the occurrence data on Alternaria toxins in agricultural commodities and feed reported
from Europe is presented in Table 12.
In Europe, approximately 300 samples of feed and agricultural commodities (for which the intended use
was not clearly indicated in the publications) were analysed for AOH and AME, and to a lesser extent
for ALT, ATX I and TeA during the last three decades.
AOH could be determined in 31 % of the samples. In sunflower seeds visibly affected by Alternaria rot,
the highest level of AOH with 1840 g/kg was observed (Logrieco et al., 1988). AOH was present in all
commodities to some extent. Levels of AOH between 130 and 340 g/kg were analysed in barley and
wheat, maize silage with visible fungal growth, oats flour for animal nutrition, feed cereals and straw
(Hggblom et al., 2007; Asam et al., 2010; Ktt et al., 2010; Rasmussen et al., 2010). Rape seed, rape
seed meal, wheat, maize and maize silage without fungal growth contained AOH concentrations
< 100 g/kg (Nawaz et al., 1997; Skarkova et al., 2005; Monbaliu et al., 2010; Rasmussen et al., 2010).
For AME, 6 % of the samples had quantifiable concentrations. The highest levels were found in feed
cereals with 184 g AME/kg (Hggblom et al., 2007) and in sunflower seeds visibly affected by
Alternaria rot with 129 g/kg (Logrieco et al., 1988). Concentrations of AME up to 100 g/kg were
present in maize, oat flour, rape seed meal and in maize silage with visible fungal growth (Nawaz et al.,
1997; Asam et al., 2010; Monbaliu et al., 2010; Rasmussen et al., 2010). No AME was detected in
wheat, rape seed samples and in maize silage without fungal growth (Skarkova et al., 2005; Monbaliu et
al., 2010; Rasmussen et al., 2010).
In Germany a north south divide could be determined for contaminations of barley and oats with AOH
and AME, as contaminations of barley occurred more frequently in the northern and those of oats in the
southern regions. For wheat no such trend could be observed (Gruber-Schley and Thalmann, 1988).
Only one study compared conventional and organic farming in relation to the concentration of
Alternaria toxins. The concentrations of AOH, AME and ALT in organic fibre flax seed and linseed
samples were higher than those from conventional farming (Krlov et al., 2006). Due to a limited
number of samples no clear conclusions on the influence of the farming system on the target toxin
incidence could be drawn.
For ALT, 164 samples were analysed, whereof 73 % showed quantifiable concentrations with the
highest level being 41 g/kg in wheat (Skarkova et al., 2005). No concentrations of ALT were found in
rape seed meal (Nawaz et al., 1997).
No traces of ATX-I were found in 30 rape seed meal samples analysed by Nawaz et al. (1997).
EFSA Journal 2011;9(10):2407
29
For TeA, 182 samples were analysed with 15 % showing quantifiable concentrations. The highest
concentration of 4310 g/kg TeA was found in oats for animal nutrition (Hggblom et al., 2007). Rape
seed meal contained 970 g/kg of TeA (Nawaz et al., 1997). No TeA was present in wheat (Skarkova et
al., 2005).
From North America some older data from the 1970s about heavily damaged sorghum report high levels
of the sum of AOH and AME between 5000 and 7900 g/kg and of ALT with concentrations up to
1500 g/kg (Seitz et al., 1975; Sauer et al., 1978). Hay, hay silage, maize meal, maize silage and mixed
feed can contain high levels of AAL-TA and AAL-TB up to 2000 and 900 g/kg, respectively (Yu et
al., 1999; Mansfield et al., 2007) (Appendix B, Table B2).
In South America some occurrence data on sunflower seed, wheat and soya bean were available. Wheat
and sunflower seed samples were investigated for AOH, AME and TeA, soya bean samples only for
AOH and AME. Measurable amounts of AOH were found in wheat with up to 1388 g/kg with low
contamination frequency, whereas sunflower seeds showed a high contamination frequency with lower
levels up to 980 g/kg. In soya bean a maximum level of 211 g/kg AOH was quantified with almost
half of the samples being contaminated. The maximum concentrations of AME were 7451 g/kg in
wheat, 1153 g/kg in soya bean and 836 g/kg in sunflower seed. TeA concentrations reached
maximum levels of 31600 and 8814 g/kg in sunflower seeds and wheat, respectively (Torres et al.,
1993; Chulze et al., 1995; Dalcero et al., 1997; Azcarate et al., 2008; Ramirez, 2011, personal
communication; Barros et al., 2011) (Appendix B, Table B2).
In Asia, the maximum levels of AME quantified were 1426 g/kg in weathered wheat from China.
AOH was present in weathered wheat with levels up to 731 g/kg. TeA was quantified with levels up to
6432 g/kg, whereas no ALT and ATX I were detected in any of the samples (Li and Yoshizawa, 2000)
(Appendix B, Table B2). The Chinese wheat samples with high levels of AOH and AME also contained
a high level of TeA. There was a significant positive correlation between the levels of AOH and AME
(r = 0.85) and between total dibenzo--pyrones (sum of AOH and AME) and TeA (r = 0.796) (Li and
Yoshizawa, 2000). AOH was also found in feed and raw materials from Russia with the highest level of
760 g/kg quantified in one silage sample (Burkin and Kononenko, 2011).
In Africa, agricultural commodities and feed were tested for AOH, AME, TeA, ALT and ATX-I. AOH
was quantified with a maximum level of 2320 g/kg in wheat, whereas it was not detected in maize,
barley, sorghum, rice, poultry feed, sunflower oil cake and cotton seed cake. For AME the maximum
levels of 2250 and 1890 g/kg were found in sorghum-based mixed feed for swine and in wheat,
respectively (Sydenham et al., 1988; Abd el-Aal, 1997). Abd el-Aal (1997) investigated different
agricultural commodities and feed. For ALT, the maximum level was found in wheat with 1480 g/kg.
Sorghum and wheat bran were not contaminated with ALT. ATX-I was only found in 7 samples with a
maximum of 1678 g/kg in wheat and 880 g/kg in poultry feed. No ATX-I was detected in maize,
barley, wheat bran, rice, cotton seed cake and sunflower oil cake (Appendix B, Table B2). TeA was
observed with a maximum concentration in wheat of 658 g/kg while barley, peanut and sunflower oil
cake contained no TeA.
In Australia, wheat and sorghum samples were analysed for AOH AME, and TeA. AOH and AME were
only present in weathered wheat in concentrations up to 1050 and 46 g/kg, respectively, and could not
be detected in undamaged wheat and sorghum. TeA was present in weathered wheat with levels up to
220 g/kg and with lower amounts also in undamaged wheat (15 g/kg) and sorghum (up to 115 g/kg)
(Webley et al., 1997) (Appendix B, Table B2).
From third countries some reported data on feed and feed raw material are available comprising mainly
wheat grain and sunflower seed (Appendix B, Table B2). Regarding import data on feed and available
occurrence feed data, sunflower seeds from South America show high levels of AOH, AME and TeA
and may lead to high exposure of animals to these Alternaria toxins. Also in wheat grain grown in
South America and Africa higher concentrations of AOH, AME, ATX-I and TeA were found than in
EFSA Journal 2011;9(10):2407
30
wheat grain grown in Europe, but wheat does not play a major role in feed imports to Europe
(EUROSTAT, 20117). As for ALT higher levels were observed in feed material from Africa than from
Europe, e.g. reported maximum concentration of ALT in wheat grain were 1480 g/kg versus 41 g/kg.
Table 12: Summary of occurrence data of Alternaria toxins in agricultural commodities and feed
reported from Europe.
Alternaria
toxin
N of total
samples
N of
positive
samples
AOH
AME
302
292
95
18
31
6
1840
184
ALT
ATX-I
TeA
164
30
182
119
0
27
73
0
15
41
n.d.
4310
Positive
samples
(%)
Maximum
concentratio
n (g/kg)
Commodity
type reported
for the
maximum
concentration
Sunflower seed
Cereals
for
animal nutrition
Wheat grain
Rape seed meal
Oats for animal
nutrition
Reference
(reporting the
maximum
concentration)
Logrieco et al. (1988)
Hggblom et al.
(2007)
Skarkova et al. (2005)
Nawaz et al. (1997)
Hggblom et al.
(2007)
AOH: alternariol; AME: alternariol monomethyl ether; ALT: altenuene; ATX-I: altertoxins I; TeA: tenuazonic acid; N:
number; n.d.: not detected
3.4.
Occurrence data in feed
No data on Alternaria toxins in feed were reported to EFSA. Occurrence data were available only from
the literature (see Appendix B, Table B2). To enable the estimation of exposure in animals the LB and
UB mean concentrations in feedstuffs were calculated from selected published papers where the
information provided made this possible (Table 13). Therefore also studies on feed from outside of
Europe were considered. The LB and UB means were calculated for each study and where more studies
were available for the same toxin and commodity the LB and UB means were calculated by weighting
for the number of samples included in each study. A total of 1150 data collected from literature was
considered in the assessment of Alternaria toxins in feed: AOH (n = 755), AME (n = 158), ALT
(n = 129) and TeA (n = 108). The results on Alternaria toxins in feed were characterised by a high
proportion of left-censored data ranging from 9 % up to 66 % for the different compounds.
http://epp.eurostat.ec.europa.eu/portal/page/portal/eurostat/home
EFSA Journal 2011;9(10):2407
31
Table 13: Mean concentration of Alternaria toxins in feeds as calculated from data reported in the literature.
Toxin
Commodity
ND
AOH
AOH
AOH
AOH
AOH
AOH
Wheat grain
Wheat grain
Wheat grain
Wheat grain
Maize grain
Maize grain
129
64
22
28
45
52
69
60
2
24
42
47
5
50
50
20
39
20
6.4
66
305
14
1.4
8.5
9.0
113
309
31
38
27
AOH
21
20
155
159
AOH
AOH
Sunflower meal and

oil cake
Sunflower seed meal
Soya bean
22
15
4
0
50
24
147
72
156
72
AOH
Soya bean
35
27
16
22
AOH
AOH
AOH
AOH
AME
AME
AME
AME
Barley grain
Wheat bran
Maize gluten
Combined feeds
Wheat grain
Wheat grain
Sunflower seed meal
Soya bean
76
10
20
216
64
22
22
15
54
6
4
157
49
1
6
0
20
20
20
20
50
50
40
48
14
14
45
13
496
423
73
363
28
26
49
28
535
425
84
363
AME
Soya bean
35
28
48
55
94
EFSA Journal 2011;9(10):2407
LOD
or
LOQ(a)
LB
mean
UB
mean
LB
mean(b)
UB
mean(b)
g/kg
50
83
5.2
32
151
158
Sample origin
Reference
Czech Republic
Argentina
China
Russia
Europe (various countries)
Russia

Azcarate et al. (2008)
Li and Yoshizawa (2000)
Burkin and Kononenko (2011)
Monbaliu et al. (2010);
Burkin and Kononenko (2011), personal
communication
Russia
EC origin, Argentina, India
Argentina
33
37
478
507
148
175
Argentina
Russia
Russia
Russia
Russia
Argentina
China
Argentina
Argentina
Nawaz et al. (1997)

Ramirez (2011), personal communication;
Barros et al. (2011)
Nawaz et al. (1997)
32
Table 13: Continued.

Toxin
Commodity
ND
ALT
TeA
TeA
TeA
Wheat grain
Wheat grain
Wheat grain
Sunflower seed meal
129
64
22
22
11
52
0
0
LOD
or
LOQ(a)
LB
mean
UB
mean
LB
mean(b)
UB
mean(b)
g/kg
5
80
100
350
19
434
2419
1900
19
499
2419
1900
942
990
Sample origin
Reference
Czech Republic
Argentina
China

Nawaz et al. (1997)
AOH: alternariol; AME: alternariol monomethyl ether; ALT: altenuene; TeA: tenuazonic acid; N: number of samples; ND: number of results below < limit of detection (LOD) or < limit of
quantification (LOQ); LB: lower-bound; UB: upper-bound; EC: European Community; (a): LOD or LOQ as considered in the calculation of LB and UB means; (b): LB and UB means
calculated by weighting for the number of samples included in each study.
EFSA Journal 2011;9(10):2407
33
3.5.
Trends in the previously reported occurrence data
In the following paragraphs the occurrence data of food, feed and agricultural commodities from Europe
reported in literature are compared with each other to look for possible trends in frequencies and
concentrations. These data may differ from the ones included in the database used in this Scientific
Opinion for exposure assessment, since some data from the literature were excluded from the database
either for not being relevant for human consumption or due to some missing information.
AOH was found more frequently in herbs, spices and condiments, grains and grain-products with
unspecified use, alcoholic beverages, fruit and vegetable juices, grains and grain-based products used as
feed than in hay and silage, grains and grain-based products for human consumption, fruit and fruit
products, oil seed meals, legumes, nuts and oilseeds, vegetables and vegetable products. However, the
highest levels of AOH were quantified in legumes, nuts and oilseeds, grains and grain-based products
with unspecified use, grains and grain-based products used as feed, hay and silage followed by oil seed
meals, vegetables and vegetable products, fruit and vegetable juices, alcoholic beverages and herbs,
spices and condiments. The lowest concentrations were found in grains and grain-based products for
human consumption and in fruit and fruit products (Table 14).
AME was found more frequently in alcoholic beverages, grains and grain products used as feed and in
legumes, nuts and oilseeds than in grains and grain products for human consumption, fruit and vegetable
juices, oil seed meals, hay and silage, vegetables and vegetable products. The highest levels of AME
were quantified in grains and grain-based products used as feed and legumes, nuts and oilseeds followed
by oil seed meals, hay and silage and vegetables and vegetable products, whereas the lowest
concentrations were observed in fruit and vegetable juices, grains and grain-based products for human
consumption and alcoholic beverages. No AME was found in fruit and fruit products and in grains and
grain-based products with unspecified use.
ALT was observed frequently in grains and grain-based products with unspecified use and sporadically
in legumes, nuts and oilseeds, both groups showing low levels of contamination. No ALT was present in
alcoholic beverages, fruit and vegetable juices, fruit and fruit products and oil seed meals.
TeA was frequently found in grains and grain-based products used as feed and in oil seed meals
followed by alcoholic beverages and grains and grain-based products for human consumption. Also the
highest concentrations were found in grains and grain-based products used as feed and in oil seed meals
followed by grains and grain-based products for human consumption and alcoholic beverages. No TeA
was quantified in fruit and vegetable juices, grains and grain-based products with unspecified use and in
vegetables and vegetable products.
ATX-I was found neither in fruit and fruit products nor in vegetables and vegetable products nor in oil
seed meals.
EFSA Journal 2011;9(10):2407
34
Table 14: Comparison of occurrence and frequency of Alternaria toxins in the different food, feed and
agricultural commodity groups(a) from Europe reported in literature.
Alternaria
toxin
Commodity
AOH
Alcoholic beverages
Grains and grain-based products for
human consumption
Grains and grain-based products used
as feed
Grains and grain-based products with
unspecified use
Hay and silage
Oil seed meals
Alcoholic beverages
human consumption
as feed
unspecified use
Hay and silage
Oil seed meals
Alcoholic beverages
unspecified use
Oil seed meals
Alcoholic beverages
human consumption
as feed
unspecified use
Oil seed meals
Oil seed meals
AME
ALT
TeA
ATX-I
Maximum
concentration
(g/kg)
20
48
6
Positive
samples
(%)
35
25
8
13
102
24
24
335
139
64
46
340
20
20
216
30
284
46
129
68
2
19
10
2
13
9
9
0
10
95
5
7
5
20
7
0
236
5
1840
81
25
0.3
2
n.d.
13
0.6
102
14
14
184
129
20
216
30
274
26
13
56
0
1
21
2
3
0
0
0
0
5
10
7
1
0
0
0
n.d.
51
129
60
5
n.d.
n.d.
n.d.
129
214
118
3
41
30
175
n.d.
N of total
samples
N of positive
samples
57
189
78
8
8
30
69
13
16
0
91
1
0
23
0
28
11
851
18
18
100
4310
129
30
266
56
30
266
0
9
0
0
0
0
0
30
0
0
0
0
n.d.
970
n.d.
n.d.
n.d.
n.d.
n.d.
(a): Commodity groups < 10 samples were not considered; AOH: alternariol; AME: alternariol monomethyl ether; ALT:
altenuene; ATX-I: ; altertoxin I; TeA: tenuazonic acid; N: number; n.d.: not detected
EFSA Journal 2011;9(10):2407
35
3.6.
Storage and processing of food and feed
Only scarce information on the stability and fate of Alternaria toxins during storage and processing of
food and feed is available.
AOH, AME and ALT were also present in tomato fruits inoculated with A. alternata after 4 weeks of
incubation at 4C (Ozcelik et al., 1990). The ability of Alternaria to grow and produce toxins at low
temperatures was also confirmed in laboratory experiments. A. alternata produced AME, TeA, ATX-I
and ATX-II in Czapek's medium at 7C, and AOH, AME and TeA in soybean-based media at 5C
(Hasan, 1995; Oviedo et al., 2009, 2010).
The possibility for spreading of Alternaria during storage of food of plant origin with high water
content and the stability of Alternaria toxins during processing conditions may result in elevated
concentrations in the end-products such as juices, sauces and purees as indicated by Terminiello et al.
(2006).
The stability of AOH, AME and ATX-I in low acid apple juice has been examined by Scott and
Kanhere (2001). It was evident that there were no apparent losses at room temperature over 20 days or
at 80C after a 20 minutes period. Furthermore, ATX-I was stable or moderately stable in apple juice
over a 1-27 days period and in a sample of red grape juice over 7 days (Scott and Kanhere, 2001).
The stability of AOH, AME and ALT was also investigated under various baking conditions, using
flour spiked with the toxins in the presence (wet baking) or absence (dry baking) of water (Siegel et al.,
2010a). Under the most realistic baking conditions (wet baking for 45-60 minutes at 200C, or for
30-45 minutes at 230C), no degradation was observed and the three toxins were recovered
quantitatively. In contrast, a pronounced degradation was observed upon dry baking. For example,
degradation after dry baking at 230C for 1 hour was about 90 % for ALT, 70 % for AOH, and 50 % for
AME. None of the degradation products found after refluxing in neutral solutions (see above) was
observed, and degradation was postulated to involve reaction with components of the flour matrix.
The effects of temperature, water activity (aw)8 and gamma irradiation on production of TeA, AOH and
ALT by A. alternata on wheat grain was investigated after 3 days of incubation. AME and ATX I were
not produced on wheat in all experiments. Concentrations of ALT were below the LOD (Table B2,
Appendix B). The optimum conditions for production of TeA and AOH were 25C and 0.98 aw. TeA
concentrations were about 4- to 8-times lower at 15C compared to the levels at 25C. There was also a
decrease of 10 to 40 % in TeA concentrations at 30C. AOH concentrations were 2- to 4-times lower at
15C compared to those at 25C. A reduction of AOH levels of 20 to 60 % was observed at 30C. The
lowest concentrations of AOH and TeA were found at 15C and 0.90 aw. Gamma irradiation at dose
levels of 1.0 to 3.0 kGy in combination with storage temperature of 15C and a water activity of 0.90 aw
decreased concentrations of AOH and TeA in wheat grain (Abd el-Aal, 1997).
Some work was done on sunflower seed processing. During the ensiling of sunflower seeds the levels of
AOH and TeA decreased as time of ensiling increased. AOH was present in all 20 samples at the
beginning of ensiling with an average concentration of 661 g/kg. A gradual decrease of AOH was
observed in 100 % of the samples at the second month and in 65 % of the samples at the fourth month of
storage. An increase of AOH levels was found in 20 % of the samples during the fourth month of
storage. No change was observed for the remaining samples. TeA was present in 80 % of the samples at
the start of ensiling with an average concentration of 16000 g/kg. A reduction in the concentrations of
TeA was found in all samples after the second and fourth month of storage with average levels of
8000 and 4500 g/kg, respectively. AME was not present in any sample at the moment of collection,
8
Water activity (aw) is defined as the ratio between the vapour pressure of the food product and the vapour pressure of distilled
water.
EFSA Journal 2011;9(10):2407
36
but was formed in one sample during storage reaching 600 g/kg at the second month and 800 g/kg at
the fourth month. Further processing of these ensiled sunflower seeds lead to meal and oil products
which were free from Alternaria toxins. (Dalcero et al., 1997). This is in contrast to the results of
Chulze et al. (1995) who found certain amounts of AOH, AME and TeA after processing of sunflower
seeds. When contaminated sunflower seeds were processed under laboratory conditions to obtain oil and
meal, different distributions of AOH, AME and TeA between the oil and the meal were observed.
Whereas AOH, AME and TeA were detected in the meal, only AME and TeA were detected in the oil.
Percentages observed in the meal were up to 93 % of AOH, 51 % of AME and 67 % of TeA of the
initial concentrations. In the oil, up to 45 % of AME, only 2 % of TeA and no AOH were found.
The effect of heat treatment on the stability of AOH, AME and TeA at 100, 115 and 121C was
evaluated in sunflower flour which is used as protein supplement to balance mixed feed rations for
poultry and pigs. During humid heat treatment at 100C the concentrations of AOH and AME remained
constant for up to 90 minutes, while TeA decreased with time to 50 % of the initial concentration after
90 minutes. When humid heat and pressure treatment were employed, the concentrations of AOH, AME
and TeA decreased. The most effective treatment in reducing AOH, AME and TeA levels was heating at
121C and 0.1 MPa for 60 minutes. Under these conditions a decontamination of 100 %, 75 % and 67 %
was observed for AME, AOH and TeA, respectively (Combina et al., 1999).
The effect of agronomic practices, weather conditions, and ensiling on the occurrence and levels of the
Alternaria toxins AAL-TA and AAL-TB was studied in maize silage in Pennsylvania (AAL-TA and
AAL-TB were not further specified into types 1 and 2). Alternaria toxins AAL-TA and AAL-TB were
found with a frequency of 23 % and 13 %, respectively. Temperature during maize development was
negatively correlated with AAL-TA occurrence and levels, while moisture conditions were positively
correlated with AAL-TA. Ensiling did not affect toxin concentration nor did agronomic practices
(tillage system, inoculant use or silo type) or silage characteristics such as dry matter, pH or organic
acid concentration (Mansfield et al., 2007).
4.
Food and feed consumption
4.1.
Food consumption
In 2010, the EFSA Comprehensive European Food Consumption Database (Comprehensive Database)
was built from existing national information on food consumption at a detailed level. Competent
authorities in the European countries provided EFSA with data from the most recent national dietary
survey in their country at the level of consumption by the individual consumer. This included food
consumption data concerning infants (2 surveys from 2 countries), toddlers (8 surveys from
8 countries), children (17 surveys from 14 ountries), adolescents (14 surveys from 12 countries), adults
(21 surveys from 20 countries), elderly (9 surveys from 9 countries) and very elderly (8 surveys from
8 countries) for a total of 32 different dietary surveys carried out in 22 different countries. Surveys on
children were mainly obtained through the Article 36 project Individual food consumption data and
exposure assessment studies for children (acronym EXPOCHI) (Huybrechts et al., in press).
Overall, the food consumption data gathered at EFSA in the Comprehensive Database are the most
complete and detailed data currently available in the EU. However, consumption data were collected by
using different methodologies and thus they are not suitable for direct country-to-country comparison.
4.1.1.
EFSA Comprehensive European Food Consumption Database
Considering the limited occurrence data available and the high proportion of left-censored data, the
CONTAM Panel decided to perform a limited dietary exposure to Alternaria toxins focussing only on
adult population. As suggested by the EFSA Working Group on Food Consumption and Exposure
(EFSA, 2011b) dietary surveys with only one day per subject were not considered for the calculation of
EFSA Journal 2011;9(10):2407
37
chronic dietary exposure, as they are not adequate to assess repeated exposure. Similarly, subjects who
participated only one day in the dietary studies although the protocol prescribed more reporting days per
individual were also excluded. Thus, food consumption data for adult population (> 18 years to
< 65 years old) were available from 14 countries (15 dietary surveys). The dietary surveys considered
for the chronic dietary exposure assessment and number of subjects in each dietary survey are presented
in Table 15. Further details on how the Comprehensive Database is used are published in the Guidance
of EFSA Use of the EFSA Comprehensive European Food Consumption Database in Exposure
Assessment (EFSA, 2011b).
Table 15: Dietary surveys considered for the chronic dietary exposure assessment and number of
subjects in the age class adults.
Country
Belgium
Czech Republic
Denmark
Finland
France
Germany
Hungary
Ireland
Italy
Latvia
Netherlands
Spain
Spain
Sweden
United Kingdom
Dietary survey(a)
Diet National 2004
SISP04
Danish_Dietary_Survey
FINDIET_2007
INCA2
National_Nutrition_Survey_II
National_Repr_Surv
NSIFCS
INRAN_SCAI_2005_06
EFSA_TEST
DNFCS_2003
AESAN
AESAN_FIAB
Riksmaten_1997_98
NDNS
Abbreviation(b)
BE/1
CZ
DK
FI/2
FR
DE/4
HU
IE
IT
LT
NL/1
ES/1
ES/2
SE/1
UK
Number of subjects
1304
1666
2822
1575
2276
10419
1074
958
2313
1306
750
410
981
1210
1724
BE: Belgium; CZ: Czech Republic; DK: Denmark; FI: Finland; FR: France; DE: Germany; HU; Hungary; IE: Ireland; IT:
Italy; LT: Latvia; NL; The Netherlands; ES: Spain; SE: Sweden; UK: United Kingdom; (a): More information on the dietary
surveys is given in the Guidance of EFSA Use of the EFSA Comprehensive European Food Consumption Database in
Exposure Assessment (EFSA, 2011b); (b): Abbreviations to be used consistently in all tables on exposure assessment.
4.2.
Feed consumption
Since consistent toxicological data for farm and companion animals are not available, with the
exception of two studies carried out in chickens (see Section 6.3.2.), only this species has been
considered for feed consumption.
In general the main feedingstuffs used in poultry diets are cereal grains, cereal by-products and
vegetable proteins. The main cereal grains used in poultry diets in Europe are wheat, maize and barley.
Other grains used to a lesser extent include rye, sorghum triticale and oats. The main vegetable protein
sources used in poultry diets are meal by-products resulting from commercial vegetable oil production,
such as soya bean, rape seed, cotton seed and sunflower meals, and legumes such as peas and lupins
(Leeson and Summers, 2008; McDonald et al., 2011). Under ad libitum feeding, daily intake increases
as the birds get older, although relative to body weight (b.w.) it declines with age. For meat producing
and egg laying birds, ad libitum feeding is widely practiced, whereas for breeding stock feed intake is
frequently restricted to maintain a steady body weight (Leeson and Summers, 2008). A feed intake of
120 g dry matter/day for a live weight of 2 kg, as reported for different poultry categories by EFSA
(2009) was used in this Scientific Opinion. The typical composition of the diets for poultry is presented
in Table 16. The composition of diets for poultry are the estimates of the CONTAM Panel, and are in
agreement with common practice (Leeson and Summers, 2008; McDonald et al., 2011).
EFSA Journal 2011;9(10):2407
38
Table 16: Typical composition of the diets for poultry (values are in %).9
Broilers
Laying hens
Wheat
Maize
38
30
38
35
Soya
bean
15
22
Lucerne
meal
4
Wheat
feed(a)
1
-
Molasses(b)
3
3
Vegetable
oil
1
2
Mineral
vitamins(b)
4
4
(a): Product of flour or malting manufacture obtained from screened grains of wheat or dehusked spelt. It consists principally
of fragments of the outer skins and of particles of grain from which less of the endosperm has been removed than in wheat
bran.10 (b): No data on concentrations of Alternaria toxins available, and therefore the contribution is assumed to be zero.
5.
Exposure assessment of Alternaria toxins in animals and humans
5.1.
Human exposure assessment
Considering the limited occurrence data available and the high proportion of the left-censored data, the
CONTAM Panel decided to perform a limited dietary exposure assessment focussing only on adults
( 18 to < 65 years old). Furthermore, due to the limited occurrence data available for the individual
Alternaria toxins and the large proportion of left-censored data in all data sets, the exposure estimates
should be regarded as being only indicative.
5.1.1.
Mean and high dietary exposure to Alternaria toxins
For calculating the chronic dietary exposure to Alternaria toxins, food consumption and body weight
data at the individual level were accessed in the Comprehensive Database. For each country, exposure
estimates were calculated per dietary survey (see Section 4.1.1). Exposure estimates were therefore
calculated for 15 different dietary surveys carried out in 14 different European countries.
The mean dietary exposure (average consumption in adult population) and the high dietary exposure
(95th percentile food consumption in adult population) to Alternaria toxins were calculated separately
for each dietary survey using consumption data recorded at the individual level. Individual food
consumption data were combined with the mean occurrence values in order to provide mean and high
percentile exposure estimates (95th percentile). Exposure estimates were calculated for both LB and UB
scenarios. The LB and UB mean concentrations of the food groups used in the exposure calculation are
presented in Table 11.
The following rules where applied in linking the occurrence data to the consumption data:
-
Broad food groups (FoodEx level 1) where all results were below LODs or LOQs were not included
in the exposure calculation (e.g. Starchy root and tubers, Non-alcoholic beverages);
Within the broad food groups, only the sub-groups (FoodEx level 2 or 3) where positive results
were reported were considered for the exposure assessment. Including all food subgroups in the
exposure calculation, regardless of whether the toxins were found or not, would have lead to
overestimation of the dietary exposure estimates in the UB scenario, in particular for TEN where
only a few food groups had results above LOD or LOQ.
Food sub-groups with less than 5 samples were not considered for the exposure calculation
regardless of whether there were quantified results or not. Overall is to note that the number of data
available for each food group (see Table 11) was limited.
There is considerable variation in poultry feeding systems throughout Europe and thus the composition of diets for poultry
does not represent average diets, nor they necessarily reflect typical feeding systems applicable to all production systems
in the Europe. However, the composition of diets is not considered to be atypical.
10
Commission Regulation (EU) No 575/2011 of June 2011 on the Catalogue of feed materials. OJ L 159, 17.6.2011, p. 25-65.
EFSA Journal 2011;9(10):2407
39
In some dietary surveys, the consumption data were not reported at the same level of detail as the
occurrence data but at an upper level. Those consumption data were linked with the occurrence data
at the upper level (see unspecified groups in Table 11).
The detailed statistics for mean and 95th percentile chronic dietary exposure to AOH, AME, TeA and
TEN (ng/kg b.w. per day) for adult population in LB and UB scenarios are presented in Table 17 and
the summary statistics in Table 18.
In the adult population, the mean chronic dietary exposure to AOH across dietary surveys ranged from
1.9 to 39 ng/kg b.w. per day. The 95th percentile dietary exposure ranged from 5.9 to 82 ng/kg b.w. per
day (Table 18). The exposure estimates for AME were lower compared to those obtained for AOH
(mean chronic dietary exposure ranged from 0.8 to 4.7 ng/kg b.w. per day; 95th percentile dietary
exposure ranged from 3.1 to 15 ng/kg b.w. per day). Considerably higher exposure estimates were
obtained for TeA. The mean chronic dietary exposure ranged from 36 to 141 ng/kg b.w. per day and the
95th percentile dietary exposure ranged from 86 to 362 ng/kg b.w. per day. The lowest exposure
estimates were calculated for TEN (mean chronic dietary exposure ranged from 0.01 to 7 ng/kg b.w. per
day; 95th percentile dietary exposure ranged from 0.00 to 13 ng/kg b.w. per day.
Although the chronic dietary exposure was not calculated for all age classes, due to the higher food
consumption per kg body weight it is expected that the dietary exposure in children might be higher
compared to adults by a factor of 2 to 3. Similarly, vegetarians might have higher exposure due to the
higher intake of food of plant origin.
Table 17: Mean and 95th percentile (P95) chronic dietary exposure to AOH, AME, TeA and TEN
(ng/kg b.w. per day) for adult population in lower-bound (LB) and upper-bound (UB) scenarios.
Dietary
survey(a)
BE/1
CZ
DK
FI/2
FR
DE/4
HU
IE
IT
LV
NL/1
ES/1
ES/2
SE/2
UK
Range of dietary exposure (LB - UB) (ng/kg b.w. per day)

AOH
Mean
P95
3.6 - 26
13 - 63
2.1 - 21 6.4 - 51
3.7 - 28
10 - 65
3.5 - 28
11 - 70
4.0 - 26
10 - 62
3.5 - 32
12 - 82
4.2 - 34
13 - 76
1.9 - 17 5.9 - 42
4.0 - 39
11 - 73
2.2 - 20
10 - 66
2.6 - 16 8.5 - 42
4.5 - 34
15 - 73
5.7 - 37
17 - 77
3.2 - 25 8.8 - 58
4.2 - 24
12 - 59
AME
Mean
P95
1.3 - 3.1
7.5 - 11
1.7 - 3.4
4.0 - 10
1.5 - 3.9
6.6 - 11
1.5 - 2.9
5.9 - 10
2 - 3.7
6.6 - 10
1.2 - 4.3
5.5 - 15
2.9 - 4.7
10 - 13
0.9 - 2.1
3.6 - 6.1
2.1 - 3.8
7.9 - 11
1 - 2.1
5.4 - 8.5
0.8 - 2.5
3.1 - 8.4
2.2 - 3.6
10 - 13
3 - 4.7
12 - 15
1.6 - 2.7
4.6 - 6.6
1.9 - 3.7
6.6 - 11
TeA
Mean
P95
41 - 94
127 - 262
64 - 125 219 - 362
46 - 91
119 - 208
56 - 129 150 - 282
36 - 78
89 - 169
46 - 93
156 - 259
69 - 111 141 - 229
59 - 110 195 - 319
58 - 141 114 - 254
39 - 71
117 - 218
36 - 72
163 - 277
53 - 104 144 - 241
55 - 107 142 - 242
40 - 78
97 - 176
49 - 97
145 - 257
TEN
Mean
0.06 - 0.09
0.7 - 1.1
0.01 - 0.02
1.2 - 5.1
0.66 - 0.93
0.26 - 0.33
6.8 7.0
0.31 - 0.4
0.17 - 0.24
1.3 - 1.4
0.23 - 0.26
0.93 - 0.98
1.4 - 1.4
0.07 - 0.08
0.34 - 0.42
P95
0.11 - 0.23
1.7 - 2.6
0(b)
4.2 - 12.1
3.1 - 3.7
0.9 - 1.2
13 - 13
0.85 - 1.11
0.5 - 0.8
1.3 - 1.9
0.6 - 0.9
1.1 - 1.2
6.4 - 6.5
0(b)
0.9 - 1.1
BE: Belgium; CZ: Czech Republic; DK: Denmark; FI: Finland; FR: France; DE: Germany; HU; Hungary; IE: Ireland; IT:
Italy; LV: Latvia; NL; The Netherlands; ES: Spain; SE: Sweden; UK: United Kingdom; b.w.: body weight; LB: lower-bound;
UB: upper-bound; P95: 95th percentile; AOH: alternariol; AME: alternariol monomethyl ether; TeA: tenuazonic acid; TEN:
tentoxin; (a): Original acronyms of the dietary surveys and the number of subjects are given in Table 15. (b): In countries
where the number of consumers of foods included in the exposure assessment were less than 5 % of the population, the 95th
percentile consumption amount was zero. Therefore the P95 dietary exposure estimates equals zero.
EFSA Journal 2011;9(10):2407
40
Table 18: Summary statistics of the mean and 95th percentile chronic dietary exposure to AOH, AME,
TeA and TEN in adult population (ng/kg b.w. per day) across the European countries.
Toxin
Summary statistics of exposure (ng/kg b.w. per day)

Minimum
LB
UB
Median
LB
UB
Maximum
LB
UB
Mean dietary exposure

AOH
AME
TeA
TEN
1.9
0.8
36
0.01
16
2.1
71
0.02
AOH
AME
TeA
TEN
5.9
3.1
89
0.00(a)
42
6.1
169
0.00(a)
3.6
26
1.6
3.6
49
97
0.34
0.42
95th percentile dietary exposure
11
6.6
142
0.90
65
11
254
1.2
5.7
3
69
6.8
39
4.7
141
7.0
17
12
219
13
82
15
362
13
AOH: alternariol; AME: alternariol monomethyl ether; TeA: tenuazonic acid; TEN: tentoxin; b.w.: body weight; LB: lowerbound; UB: upper-bound; (a): The 95th percentile estimates equals zero because less than 5 % of subjects reported consumption
of foods contributing to the TEN exposure.
5.1.1.1. Contributions of different food groups to exposure of Alternaria toxins

The contribution of individual food groups to dietary exposure to AOH, AME, TeA and TEN varied
between the dietary surveys. This is explained by the specific food consumption patterns in the
individual European countries and even in the different regions of one country. The contribution of the
individual food groups to the dietary exposure to AOH, AME, TeA and TEN was calculated for both
LB and UB scenarios. A summary of the median values calculated from the average contribution of
each food group across the dietary surveys and the range of the lowest and highest average contribution
is shown in Table 19.
For AOH, the largest contribution to the dietary exposure was made by Grains and grain-based
products mainly due to the high consumption of foods included in this group. Other important
contributors were Fruit and fruit products, Fruit and vegetable juices and Alcoholic beverages
(wine). Vegetables and vegetable products contributed to a less extent to the dietary exposure and is
mainly related to the consumption of tomato products. Although the mean concentration of AOH in
Oilseeds and Vegetable oils, in particular in sunflower seeds and sunflower oil was relatively high,
due to the low consumption of these foods their contribution to the dietary exposure was less important.
In general, a similar pattern was observed in the contribution of most of the food groups to the dietary
exposure to AME with the exception of Vegetable oils and Fruit and fruit products which had a
higher contribution to the exposure to AME in both LB and UB scenarios. Conversely, the contribution
of Fruit and vegetable juices and Alcoholic beverages was lower but only in the LB scenario.
For TeA, the most important contributors were Vegetables and vegetable products, Grains and grain
products and Alcoholic beverages (beer). Oilseeds and Vegetable oils had an important
contribution only in dietary surveys where the consumption of sunflower oil and sunflower seeds was
the highest.
The exposure to TEN was mainly linked to the consumption of sunflower oil and sunflower seeds, these
foods being the only contributors to the dietary exposure. Grains and grain products and Vegetables
and vegetable products had a significantly lower contribution.
EFSA Journal 2011;9(10):2407
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In conclusion, there is no one food group to be considered as the major contributor to the dietary
exposure to Alternaria toxins. Depending on the toxin and the food consumption pattern in different
countries, the contribution is mainly made by Grain and grain-based products, Vegetable and
vegetable products, Fruits and fruit products, Fruit and vegetable juices, Alcoholic beverages,
Vegetable oils and Oilseeds. However, Alternaria toxins were reported only in a limited number of
food items in each of these food groups and therefore not all foods in the group contribute to the dietary
exposure.
Table 19: Contribution (%) of the different food groups to chronic dietary exposure to AOH, AME,
TeA and TEN in adult population in lower-bound and upper-bound scenarios. Median values across
dietary surveys and range of the average contribution are presented.
Food group
AOH
AME
TeA
TEN
Median contribution across dietary surveys

(Lowest contribution Highest contribution)(a)
Lower-bound
35
(19 - 69)
29
(20 - 77)
8.9
(4.5 - 28)
0.10
2.5
(0.23 - 17)
2.0
(0.04 - 26)
41
(14 - 68)
0.20 (0.0 - 2.4)
Oilseeds
(0 - 33)
2.4
(0.0 - 25)
1.6 (0.0 - 20.0)
44
(0 - 13)
6.2
(0.0 - 42)
2.6 (0.0 - 57.8)
55 (0.0 - 100)
2.1
0.7
1
19
(5.9 - 35)
39
(7.6 - 75)
4.0 (0.0 - 11.5)
0.0 (0.0 - 0.0)
19
(0.4 - 44)
0.7 (0.03 - 4.1)
0.60
(0.0 - 8.5)
0.0 (0.0 - 0.0)
Alcoholic beverages
12
(1.8 - 25)
1.2 (0.18 - 2.8)
33
(6.8 - 62)
0.0 (0.0 - 0.0)
0.0
(0.0 - 0.0)
0.0
(0.0 - 0.0)
0.0
(0.0 - 0.0)
0.0 (0.0 - 0.0)
42
(24 - 70)
22
(12 - 67)
25
(15 - 52)
0.90
(0.0 - 79)
(0.2 - 8.8)
2.3
(0.2 - 15)
28
(12 - 48)
0.90
(0.0 - 24)
(0.0 - 15)
1.1
(0.0 - 12)
0.80
(0.0 - 11)
33
(0.0 - 98)
Vegetable oils
2.0
0.8
1
1.4
(0.0 - 21)
4.0
(0.0 - 42)
1.5
(0.0 - 36)
54
(0.0 - 99)
14
(3.6 - 31)
21
(4.3 - 49)
5.3 (0.50 - 12)
0.0 (0.0 - 0.0)
19
(0.54 - 49)
20
(0.5 - 52)
1.5 (0.10 - 16)
0.0 (0.0 - 0.0)
Alcoholic beverages
9.7
(1.9 - 23)
12
(2.1 - 27)
23
(4.3 - 50)
0.0 (0.0 - 0.0)
0.0
(0.0 - 0.0)
0.0
(0.0 - 0.0)
0.0
(0.0 - 0.0)
0.0 (0.0 - 0.0)
Vegetable oils
(0.0 - 23)
(0.0 - 98)
Upper-bound
Oilseeds
AOH: alternariol; AME: alternariol monomethyl ether; TeA: tenuazonic acid; TEN: tentoxin; (a): The range refers to the
lowest and highest average contribution calculated for the individual dietary survey.
5.2.
Estimation of intake Alternaria toxins in feed by farm livestock
Due to the fact that toxicity data suitable for risk assessment of Alternaria toxins have only been
reported for chicken, estimation of intake of Alternaria toxins in feed by farm livestock was limited to
this species. Broiler and laying hen exposure have been estimated from the feed intake data as described
in Section 4.2. and the mean LB and UB concentrations calculated from the occurrence data reported in
the literature (Table 13). Considering the lack of information on the levels of toxins other than AOH in
the different feed commodities, the intake estimation taking into account LB and UB values was only
based on this compound. For broilers (2 kg), LB and UB exposure to AOH would be 3.1 and
EFSA Journal 2011;9(10):2407
42
6.0 g/day, respectively, whereas for laying hens (2 kg) the values would be 2.9 and 5.3 g/day. The
exposure estimate should be regarded as being only indicative.
6.
Hazard identification and characterisation
6.1.
Toxicokinetics: animal and in vitro studies
The only published study on the toxicokinetics of Alternaria toxins in laboratory animals is a report on
the distribution, elimination and metabolism of AME in adult male Sprague-Dawley rats (Pollock et al.,
1982a). 14C-Labelled AME of low specific radioactivity was obtained from A. solani Sorauer grown in
the presence of 1,2-14C-acetate. When administered in olive oil by gavage at a dose of 0.25 mmol/kg
b.w., 81-87 % of the administered radioactivity were recovered from the feces within three days and
shown to be unchanged AME. Only 5-9 % of the dose were excreted with the urine, mostly on day 1
and as polar metabolites. Levels of radioactivity in various tissues were very low. The authors conclude
that AME is poorly absorbed from the gastrointestinal tract, but the absorbed proportion is extensively
metabolised and does not persist in tissues. No other studies on the fate of Alternaria toxins in vivo were
identified.
Although this study has analytical limitations, the conclusion of poor absorption and extensive
metabolism of AME is consistent with more recent in vitro studies. Pfeiffer et al. (2007b) have observed
that 7 oxidative metabolites were formed from AME upon incubation with NADPH-fortified rat liver
microsomes. In addition to O-demethylation to AOH, monohydroxylation occurred at each of the
4 aromatic positions of rings A and C and also at the 1-methyl group of AME (see Section 1.3, Figure
1). Likewise, AOH gave rise to the corresponding microsomal metabolites. The same metabolites of
AME and AOH were formed with microsomes from porcine or human liver. As the products of
aromatic hydroxylation of AME or AOH constitute catechols or hydroquinones, the oxidative
metabolism of these Alternaria toxins may be of toxicological relevance. When ALT, which has an
aromatic ring A and an aliphatic ring C (see Figure 1), were incubated with rat, porcine and human
hepatic microsomes/NADPH, the major metabolite was the catechol formed through hydroxylation at
C-8 (Pfeiffer et al., 2009).
Pfeiffer et al. (2008) analysed 12 isoforms of human cytochrome P450 (hCYP), commonly expressed in
various tissues, for their activity to hydroxylate AME, AOH, ALT or iso-ALT. The most active
monooxygenase for AME and AOH was hCYP1A1, and lower activities were observed for hCYP1A2,
2C19 and 3A4. In contrast, ALT was preferentially hydroxylated by hCYP2C19, followed by 2C9 and
2D6, and the dominating metabolite was the 8-hydroxylated catechol. From the activities of the hCYP
isoforms, significant extrahepatic hydroxylation must be expected, e.g. in the lung and esophagus for
AME and AOH, and in the intestine for ALT.
As AME and AOH have 2 and 3 phenolic hydroxyl groups, respectively, the formation of conjugated
metabolites must be expected. No in vivo data are yet available. However, when AME and AOH were
incubated with UDPGA-fortified hepatic and intestinal microsomes from rats, pigs and humans, AME
was predominantly converted to its 3-O-glucuronide, whereas AOH gave rise to two major conjugates
identified as AOH-3-O-glucuronide and AOH-9-O-glucuronide (Pfeiffer et al., 2009). In the same
study, the activities of 10 recombinant human UDP-glucuronosyltransferases (hUGTs) were determined.
Eight and 9 hUGT isoforms had significant activities for AME and AOH, respectively, suggesting that
both Alternaria toxins are readily glucuronidated in hepatic and extrahepatic tissues.
The formation of AME-3-O-glucuronide and of both AOH-3-O-glucuronide and AOH-9-O-glucuronide
have been observed when the respective toxins were incubated with differentiated Caco-2 cells
(Burkhardt et al., 2009). Although derived from a colon carcinoma, differentiated Caco-2 cells have
many features in common with human intestinal epithelial cells. They are very low in CYP activity but
EFSA Journal 2011;9(10):2407
43
have active UGTs and sulfotransferases. Accordingly, the 3-O-sulfates of AME and AOH were also
identified as conjugated metabolites in these cells (Burkhardt et al., 2009).
Differentiated Caco-2 cells grown in inserts of cell culture wells can be used as an in vitro-model for
intestinal absorption. The absorption of AOH and AME have been studied in this system and the
apparent permeability coefficients determined (Burkhardt et al., 2009). According to these data, AOH
can be expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the
portal blood both as aglycone and as glucuronide and sulfate. In contrast, intestinal absorption of AME
appears to be poor and limited with no AME aglycone and only AME conjugates reaching the portal
blood.
The rapid metabolic conversion of AME and AOH to their glucuronides and sulfates raises the question
of whether hydroxylation of these toxins occurs in the presence of competing conjugation reactions,
especially those occurring in the gastrointestinal tract subsequent to absorption. In order to address this
question, precision-cut rat liver slices were used, which are an established model for metabolic studies
under in vivo-like conditions. When AOH or AME were incubated with rat liver slices, 4 catechol
metabolites of AOH and 2 of AME, together with several of their O-methylation products were clearly
identified (Burkhardt et al., 2011). The methylation of catechols by catechol-O-methyl transferase is an
expected metabolic pathway. The hydroxylated and methylated AOH and AME metabolites were
predominantly present as conjugates with glucuronic acid and/or sulphate (Burkhardt et al., 2011). In
preliminary studies with bile duct-cannulated male rats dosed with AOH by gavage, the 4 catechol
metabolites or their O-methyl ethers could also be demonstrated (Burkhardt et al., 2011). This study
clearly shows that AOH and AME undergo catechol formation in vivo. Catechols are suspected to form
reactive intermediates such as quinones and semiquinones, resulting in subsequent DNA adducts and
production of reactive oxygen species. However, this mechanism has yet to be confirmed for AOH and
AME.
CYP-mediated metabolism has also been reported for TEN in vitro (Delaforge et al., 1997). TEN
labelled with 14C in the N-methyl group of the ,-dehydrophenylalanyl moiety (see Figure 1) was used
in this study. Upon incubation with rat liver microsomes/NADPH, partial cis/trans-isomerisation of
dehydrophenylalanine gave rise to iso-TEN. The major metabolites, identified by combined use of the
14
C-label detection, UV spectroscopy and MS, were the N-demethylation products arising from
oxidative N-demethylation of TEN and iso-TEN at the alanine moiety. Small amounts of metabolites,
resulting in the loss of the 14C-label, resulted from N-demethylation at the dehydrophenylalanyl moiety
and of both N-methyl groups (see Figure 1). Moreover, small quantities of 4 monohydroxylation
products of TEN and/or iso-TEN were observed in the microsomal incubations but were not further
characterised. The interaction of CYP with TEN was also demonstrated by a type I binding spectrum
indicating binding to the active site of the enzyme. The spectral interaction and formation of oxidative
metabolites was most pronounced with hepatic microsomes from dexamethasone-treated rats,
suggesting that TEN metabolism is preferentially mediated by CYP3A isoforms. When several human
CYP isoforms expressed in yeast were compared with respect to oxidative TEN metabolism, hCYP3A4
was the most active isoform and hCYP3A5 ranked second with half the activity of 3A4; hCYP2C8 had
low activity and hCYP1A1, 1A2, 2C9, 2C18 and 2D6 were inactive (Delaforge et al., 1997).
No studies were identified addressing the carry over of secondary plant metabolites present in
Alternaria spp.
6.2.
In vitro toxicity
6.2.1.
Cytotoxicity
There is a high degree of heterogeneity in the susceptibility of mammalian cell lines to the toxic effects
of Alternaria toxins. In the human cancer HeLa cell line, the most cytotoxic was found to be ATX-II,
EFSA Journal 2011;9(10):2407
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followed by AOH as measured by inhibition of cell replication in the human cancer HeLa cell line (Pero
et al., 1973). ATX-II was also found to be the most cytotoxic in the Chinese V79 hamster lung
fibroblast cell line by using a colony survival assay and ability to inhibit gap junction communication
(Boutin et al., 1989), However, toxicity was 100-fold higher than in the HeLa cell line. In V79 cells
treated with AOH, an arrest of cell cycle in the G2 and S-phases was reported (Lehmann et al., 2005). A
significant reduction of cells in the S phase together with the arrest of the cells in the G0/G1 phase was
reported in porcine endometrial cells after exposure to AOH (Wollenhaupt et al., 2008). The addition of
the AAL-TA1 to African green monkey kidney cells (CV-1) was reported to induce the stereotypical
hallmarks of apoptosis, including the formation of DNA ladders, compaction of nuclear DNA, and the
subsequent appearance of apoptotic bodies (Wang et al., 1996).
6.2.1.1.
Mechanism of cytotoxicity
The mechanism of cell killing is very different depending on the type of Alternaria toxin. The principle
mode of action of TeA appears to be the inhibition of protein synthesis by suppression of the release of
newly formed proteins from the ribosomes (Davis et al., 1977). TeA is is the first toxin from a
phytopathogen reported as a natural photosynthesis inhibitor with several action sites, which mainly
interrupts photosystem II electron transport (Chen et al., 2007, 2010). Whether mitochondria are a
possible target of TeA induced toxicity in mammalian cells has not been addressed yet.
AAL-toxins are structurally related to sphinganine, a precursor in sphingolipid biosynthesis. Fusarium
moniliforme toxins (fumonisins) are also members of sphinganine analogue mycotoxins. Structurally,
these mycotoxins resemble the sphingoid bases, sphingosine and sphinganine, that are reported to play
critical roles in cell communication and signal transduction. Their major mechanism of toxicity in
animal cells is the disruption of the sphingolipid biosynthesis by competitively inhibiting the ratelimiting enzyme, ceramide synthase. Besides their role as structural components of biomembranes,
sphingolipids can be determinants in the proliferation or death of cells, depending on the type of cells
studied. The exposure of rat primary hepatocytes to AAL-TA and AAL-TB in culture was shown to
increase significantly the sphinganine concentration as measured by HPLC (van der Westhuizen et al.,
1998). This mechanism of action is shared with fumonisin B1, which is hepatotoxic, nephrotoxic and
carcinogenic in animals (reviewed in Stockmann-Juvala and Savolainen, 2008).
6.2.2.
Genotoxicity
There are no genotoxicity data available for Alternaria toxins in vivo.

6.2.2.1. Bacterial cells
AOH and AME were reported as not or weakly mutagenic in S. typhimurium TA98 and TA100 (Scott
and Stoltz, 1980; Davis and Stack, 1994) but AOH was shown to induce mutations in TA102 strain with
or without metabolic activation demonstrating direct-acting AT base pair mutagenicity (Schrader et al.,
2006). AOH and AME were both strongly mutagenic in Bacillus subtilis rec assay (Kada et al., 1984)
and Escherichia coli ND160 reverse mutation assay (An et al., 1989; Zhen et al., 1991) which measures
frameshift mutations. ATX-I, ATX-II and ATX-III caused gene mutations in TA98, TA100 and
TA1537 strains with or without metabolic activation in the following order ATX-III > ATX-II > ATX-I.
The potency of ATX-III was 10-fold lower than that of the mycotoxin aflatoxin B1, which is highly
mutagenic and a well established human hepatocarcinogen (Stack et al.,1986; Stack and Prival., 1986).
Nitrosylation of ATX-I generated a potent direct-acting frameshift mutagen at C sites in TA97 strain
(Schrader et al., 2006). TeA and TEN were reported as non mutagenic in TA97, TA98, TA100, TA102
and TA104 strains with or without metabolic activation and nitrosylation did not increase mutagenicity
(Schrader et al., 2006).
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6.2.2.2. Mammalian cells

It was reported that AOH induced DNA single-strand breaks in closed circular double stranded
supercoiled DNA in a cell-free system (Xu et al., 1996) and in cultured primary rat hepatocytes (Liu et
al., 1982). AOH and AME (5 to 50 M, 1 hour) caused a concentration-dependent induction of DNA
strand breaks in cultured Chinese hamster V79 cells, human liver HepG2 cells and human colon
HT-29 cells (Pfeiffer et al., 2007a). After treatment for 24 hours, DNA strand breaks were observed in
HepG2 but not HT-29 cells. Analysis of the 24 hour-incubation media of HT-29 cells showed that both
toxins were completely glucuronidated, whereas 75 % were still present as unconjugated compounds in
the 24 hour-media of HepG2 cells, suggesting that glucuronidation eliminates the DNA strand breaking
potential of AOH and AME. AOH induced kinetochore-negative micronuclei in Ishikawa and V79 cells
(Lehmann et al., 2006). Various studies reviewed in Liu et al. (1991) showed that the fungal extract of
A. alternata besides inducing unscheduled DNA synthesis in cultured human amniotic cells,
chromosomal aberrations and sister chromatid exchanges in human peripheral blood lymphocytes
increases significantly mutations at the HPRT gene locus in Chinese hamster V79 cells, and induces
transformation of NIH/3T3 mouse fibroblast cells. AME was isolated from the extract and shown to
induce HPRT mutations in cultured V79 cells as well as transformation of NIH/3T3 mouse fibroblast
cells. ATX-I and ATX-III were shown to induce transformation of C3H/10T1/2 murine fibroblasts and
activated the EBV-early antigen in Raji cells (Osborne et al., 1988). Nitrosylated ATX-I was reported to
be mutagenic in V79 cells (Schrader et al., 2006). Both AME and AOH induced mutations of the Ha-ras
gene in cultured human fetal esophageal epithelium after treatment for 4 hours (Dong et al., 1993).
Furthermore, Brugger et al. (2006) have demonstrated that AOH (10 M) gave rise to a significant and
concentration-dependent induction of HPRT and TK mutations in cultured Chinese hamster V79 cells
and in mouse lymphoma L5178Y tk+/ cells, respectively. The mutagenic potency of AOH was about
50-fold lower than that of the established mutagen 4-nitroquinoline-N-oxide in both cell lines.
Discrimination between small and large colonies in the TK assay revealed the predominant induction of
small colonies, which are indicative for extensive chromosomal deletions and which correlated with the
induction of micronuclei in mouse lymphoma cells.
6.2.2.3. Mechanism of genotoxicity
AOH has been recently characterised as an inhibitor of DNA topoisomerase I and II with a certain
selectivity for the II alpha isoform (Fehr et al., 2009). DNA topoisomerases are enzymes responsible for
regulation of genomic DNA supercoiling and participate in essential processes of cells such as
replication, transcription, recombination, repair, etc. Since this effect occurred within the same
concentration range as DNA breakage induction, it has been proposed that inhibition of topoisomerase I
and II might at least contribute to the genotoxic properties of AOH. The interference of AOH with
topoisomerase I and II is not limited to the inhibition of the catalytic activity but it was also found to
stabilise the covalent topoisomerase-DNA intermediates and here, the II -isoform was preferentially
targeted. The collision of the stabilised complex with the replication forks leads to double strand breaks
thus providing a plausible mechanism for the clastogenic effects of AOH. As expected by a
topoisomerase inhibitor the human tyrosyl-DNA phosphodiesterase I (TDP1), a key enzyme in the
repair of covalent DNA-topoisomerase adducts, affects AOH-induced genotoxicity. Human carcinoma
cells overexpressing this enzyme presented significantly less DNA breakage than cells expressing a
mutant inactive form of TDP1 while damage was increased in TDP1-suppressed cells (Fehr et al.,
2010).
In conclusion, in vitro data provide clear evidence of the genotoxicity of some Alternaria toxins in
bacteria and mammalian cells. Mutagenic and clastogenic events, induction of DNA breaks as well as a
potential role in cell transformation have been described for AOH and AME in several mammalian cell
systems. Inhibition of DNA topoisomerases, which are necessary for normal functioning of the cells as
well as production of oxidative damage, might account for these effects. If AOH genotoxicity is
confirmed to be mostly exerted via inhibition of DNA topoisomerases (i.e. by stabilisation of the
EFSA Journal 2011;9(10):2407
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cleavage complex, which, in turn, may result in the formation of a DNA strand break) this indirect
mechanism would involve the concept of a threshold for the genotoxic effects.
6.3.
Toxicity in animals
6.3.1.
Laboratory animals and bioassays
6.3.1.1. Acute toxicity

It is known that Alternaria isolates grown in laboratory culture are toxic to chickens, rats, chicken
embryos (Ostry, 2008). Intraperitoneal (i.p.) injection at a dose of 100 mg/kg per day for 3 consecutive
days of partially purified crude Alternaria extracts is lethal in mice (Pero et al., 1973).
The toxicity data on the purified Alternaria toxins are limited. ALT was the most toxic dibenzo-pyrone when given in a single dose, administered i.p., to mice. In mice the LD50 (i.p.) values of AME
and AOH are greater than 400 mg/kg of b.w. (Table 20). That of ALT is lower with 1/3 mice dying at
50 mg/kg b.w. (Pero et al., 1973; Visconti and Sibilia, 1994). The animals receiving AOH and AME
showed occasional gastric spasms and periodic panting (Pero et al., 1973). In the Chicken embryo assay
AOH, AME and ALT caused no mortality at doses up to 1000, 500 and 1000 g per egg, respectively
(Griffin and Chu, 1983).
A dose of 200 mg/kg b.w. of the Alternaria toxins ATX-I and ATX-II is lethal to mice. These toxins
cause inactivity and subendocardial and subarachnoid haemorrhaging. On autopsy, blood is found in the
cerebral ventricles (Pero et al., 1973).
The LD50 of TeA administered to mice by different routes of dosing is between 125-225 and
81-115 mg/kg b.w., for males and females, respectively (Table 21). Toxic symptoms included diarrhoea,
muscle tremor and convulsion (Woodey and Chu, 1992; Botallico and Logrieco, 1998). In the study of
Smith et al. (1968) the intravenous (i.v.) LD50 values of sodium tenuazonate were 162 and 115 mg/kg
b.w. for male and female mice respectively, and the oral LD50s were 186 mg/kg b.w. for males and
81 mg/kg b.w. for females. In rats the reported i.v. LD50s for sodium tenuazonate are 146 and 157 mg/kg
b.w. for male and female rats respectively, and the oral LD50 doses are 180 and 168 mg/kg b.w.
respectively (Smith et al., 1968). In the chicken embryo assay TeA caused embryonal death with an
LD50 of 548 g per egg (Griffin and Chu, 1983). Assuming an egg volume of 50 mL and uniform
distribution of TeA this corresponds to approximately 10 g/mL of TeA.
Purified AAL-toxins were given to rats by intragastric administration of a single dose (100 mg/kg b.w.)
without causing toxic effects (Mirocha et al., 1992).
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Table 20: Acute toxicity of Alternaria toxins in mice (intraperitoneal administration).

Compound
AOH(a)
Mouse strain
DBA/2
AME(a)
DBA/2
AOH +AME(a)
ALT(a)
ATX-I(a)
DBA/2
DBA/2
CD-1
ATX-II(a)
CD-1
TeA(b)
ICR
Dose (mg/kg b.w.)

100
200
400
100
200
400
100 + 100
50
100
200
100
200
Lethality
2/10
3/10
3/10
0/10
0/10
1/10
3/10
1/3
0/8
8/8
0/2
2/2
LD50 (mg/kg b.w.)
150
b.w.: body weight; i.p.: intraperitoneal; (a): female mice, Pero et al. (1973); (b) male mice, Woodey and Chu (1992).
Table 21: LD50 values of TeA.

Species
Mouse
Route of
administration
i.v.(a)
LD50
(mg/kg b.w.)
162 (male)
115 (female)
Mouse
oral(a)
186 (male)
81 (female)
Smith et al. (1968)
Mouse (ICR)
i.v.
i.p.
s.c.
oral
125 (male)
150 (male)
145 (male)
225 (male)
Woodey and Chu (1992)
Rat
i.v.(a)
146 (male)
157 (female)
Smith et al. (1968)
oral(a)
180 (male)
168 (female)
Smith et al. (1968)
0.548(b)
Griffin and Chu (1983)
Chicken embryo
Reference
Smith et al. (1968)
b.w.: body weight; i.v.: intravenous; i.p.: intraperitoneal; s.c.: subcutaneous.

(a): Sodium salt of TeA; (b): mg per egg (approximately 10 g/mL of TeA).
6.3.1.2. Short term toxicity

No evidence of toxicity was observed in rats when AME, AOH and ALT were fed for 21 days at levels
up to 24, 39 and 10 mg/kg feed, respectively (Sauer et al., 1978).
Two monkeys, one male and one female, were given repeated oral doses of TeA (Smith et al., 1968).
The treatment was initially with 22.4 mg/kg b.w. for 21 days. As no untoward reaction occurred the
dose was increased to 48.8 mg/kg b.w. on day 22. No adverse effects were seen for the next 9 days and
the dose was increased again to 89.6 mg/kg b.w. on day 33. This produced vomiting in both animals.
One animal had bloody diarrhoea after two treatments and then became moribund. Necropsy showed
hemorrhagic gastroenteropathy. The other monkey tolerated treatment for 15 consecutive days, although
usually vomited after treatment.
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6.3.1.3. Reproductive and developmental toxicity

The data on the developmental toxicity of Alternaria toxins are limited. AME was maternally toxic and
fetotoxic (as demonstrated by the increased number of resorptions and decreased foetal weights) to
Syrian golden hamsters when given i.p. at a single dose of 200 mg/kg b.w. on day 8 of gestation
(Pollock et al., 1982b) (Table 22). No toxic effects were seen at dose levels of 50 or 100 mg/kg b.w.
(single doses). In the study of Pero et al. (1973) DBA/2 mice were administered test compounds either
subcutaneously (s.c.) in dimethylsulphoxide (DMSO) or orally in honey:water (1:1) from days 9-12 or
13-16 of gestation. The mice were sacrificed for examination of the young on day 20. A fetotoxic effect
of AOH (dead and resorbed fetuses/litter and runts/litter) was found following a s.c. dose of 100 mg/kg
b.w. from days 9-12 of gestation, administered s.c. in DMSO (Pero et al., 1973). No effect was seen at
50 mg/kg b.w. When administered from days 13 to 16 the dose of 100 mg/kg b.w. gave an increase in
malformed fetuses/litter. No effect was seen at 25 or 50 mg/kg b.w. A synergistic fetotoxic effect was
caused by AOH and AME (1:1) with a dose of 25 mg/kg b.w. each (s.c., in DMSO, days 9-12 of
gestation) (Pero et al., 1973). There was also an increase in malformed fetuses/litter, although this was
not significant. There is no report of this treatment being carried out orally. AME administered orally at
50 mg/kg b.w. in honey-water (1:1) did not produce a statistically significant increase in fetotoxicity or
malformed foetuses. The influence of AOH, AME, TeA and ALT on chicken embryonic development
was determined by Griffin and Chu (1983). AOH, AME and ALT caused no teratogenic effect at doses
up to 1000, 500 and 1000 g per egg, respectively. TeA was tested over the range 150-1500 g per egg
(approximately 3-30 g/mL) and no teratogenic effect was elicited at either lethal or sublethal dose
levels (Griffin and Chu, 1983).
Table 22: Reproductive and developmental toxicity of Alternaria toxins (lowest-observed-adverseeffect level (LOAEL)).
Compound
Species
AME
Syrian golden
hamster
AOH
AME+AOH
Dose
(mg/kg b.w.)
200
Route
Toxic effect
Reference
i.p. day 8 of
gestation
Pollock et al.
(1982b)
Mouse (DBA/2)
100
s.c., daily doses,

days 9-12 of
gestation
Mouse (DBA/2)
100
Mouse (DBA/2)
25 (of each
compound)
s.c, daily doses,

days 13-16 of
gestation
s.c., daily doses,
days 9-12 of
gestation
Maternally toxic.
Increased
resorptions and
decreased fetal
weights.
Increased dead
and resorbed
fetuses/litter, and
runts/litter
Increased
malformed
foetuses/litter
Increased dead
and resorbed
fetuses/litter, and
runts/litter
Pero et al. (1973)
Pero et al. (1973)

Pero et al. (1973)
b.w.: body weight; i.p.: intraperitoneal; s.c.: subcutaneous.
6.3.1.4. Mechanism of reproductive and developmental toxicity

AOH is a diphenolic compound structurally similar to certain estrogen-mimicking substances. AOH was
shown to bind to cell-free recombinant human estrogen receptor (ER) and and induced the activity
and the expression of the alkaline phosphatase in a human endometrial adenocarcinoma cell line
(Ishikawa cells) (Lehmann et al., 2006). AOH functioned as a pure ER antagonist, although the
estrogenicity of AOH was about 0.01 % of that of the endogenous hormone estradiol. A more recent
study (Wollenhaupt et al., 2008) was unable to confirm an influence of AOH on ER in porcine
EFSA Journal 2011;9(10):2407
49
endometrial cells. Cell type-specific sensitivity to AOH (e.g. different receptors) might explain these
contradictory results. An effect of AOH on regulator proteins of cap-dependent translation has also been
described (Wollenhaupt et al., 2008) in porcine endometrial cells indicating that the toxic effect of AOH
could also be exerted at translational levels.
Tiemann et al. (2009) have reported that AOH and AME, but not TeA, decreased progesterone (P4)
synthesis in porcine ovarian cells in vitro. P4 is an important ovarian hormone involved in preparing the
reproductive tract for zygote implantation and maintaining pregnancy. Already 0.8 M, AOH and AME
inhibited P4 secretion. During P4 biosynthesis, an important enzymatic step is the conversion of
cholesterol to pregnenolone by the mitochondrial cytochrome P450 cholesterol side-chain cleavage
enzyme (P450scc). The abundance of P450scc protein was already significantly reduced by 0.8 M
AOH and AME. In view of the fact that granulosa cells directly influence the metabolic and structural
growth of the oocytes, exposure to AOH and AME may affect reproductive performance by interfering
with follicular development in swine and possibly other mammalian species.
6.3.1.5. Carcinogenicity
Precancerous changes were reported in oesophageal mucosa of mice (groups of 10 animals) fed in the
drinking water with 50-100 mg/kg b.w. per day of AME or 25 mg/kg b.w. per day of TeA for 10 months
(Yekeler et al., 2001), suggesting the possibility that progression to oesophageal cancer might occur
after prolonged exposure. The highest number of precursor lesions on oesophageal mucosa determined
by the authors was seen with TeA followed by AME at 100 mg/kg b.w. per day and then AME at
50 mg/kg b.w. per day. However, the CONTAM Panel considered questionable the interpretation of the
figures showing the histopathologic oesophageal lesions, and, on the basis of the images provided, was
not fully convinced by the presence of dysplastic changes in the oesophageal mucosa.
There is also some evidence of carcinogenic properties of AOH, although the assays were nonconventional. Tissue blocks of the oesophagus of human embryo were cultured for one week and then
treated with AOH for 24 hours and cultured for a further 2 weeks. The blocks were then implanted into
nude mice (BALB/c), and squamous cell carcinoma developed in 1 of the 3 animals (Liu et al., 1992).
No figures were shown, and this study could not be interpreted by the CONTAM Panel. No cancer
studies of these Alternaria toxins in experimental animals have been carried out.
Liu et al. (1991) reported that A. alternata contamination in regions where high oesophageal cancer
morbidity is observed was higher than in regions with low oesophageal cancer morbidity, and suggested
that the higher incidence of mutagenic Alternaria toxins in grains in high oesophageal cancer regions
such as Linxian in China might be a responsible factor for the cancer in those regions (Liu et al., 1991).
However, grains from counties of high incidence of oesophageal cancer were also infected by other
toxigenic fungi such as Penicillium cyclopidium, Fusarium moniliforme, Aspergillus nidulans and
Aspergillus fumigatus, and the contamination rates were all higher than those in areas of low incidence
of oesophageal cancer. In these conditions a clear association between any particular A. alternata
mycotoxin with this increased cancer incidence cannot be established.
6.3.2.
Farm and companion animals
Although it is known that Alternaria isolates grown in laboratory culture are toxic to chicken (Ostry,
2008), the available data on the toxicity of purified Alternaria mycotoxins are scarce. No evidence on
toxicity was observed in chicks when AME, AOH and ALT were fed for 21 days at the levels up to 24,
39 and 10 mg/kg feed, respectively (Sauer et al., 1978).
In day-old chicks fed with AME in the diet at levels up to 100 mg/kg feed for four weeks there was no
loss of performance or mortality (Griffin and Chu, 1983). The reported LD50 of TeA for day-old chicks
is 37.5 mg/kg b.w. (oral) (Giambrone et al., 1978). TeA given in the diet at 10 mg/kg of feed or by daily
EFSA Journal 2011;9(10):2407
50
oesophageal intubation at the levels of 1.25 or 2.5 mg/kg b.w. to 3-week old broilers for 3 weeks
resulted in decreased weight gain and lowered feed efficiency during the second and third week of toxin
administration. Although no mortality or morbidity was noted in chickens from the 3 groups, marked
macroscopic and microscopic lesions were observed, including enlarged and mottled spleen,
haemorrhage in the intestinal lumen and in thigh muscle (Giambrone et al., 1978).
Two beagle dogs (male and female) received TeA orally at 10 mg/kg b.w. per day (gelatine capsules,
4 separate doses of 2.5 mg/kg b.w.) for 8-9 days. The animals became moribund by days 8 and 9 (Smith
et al., 1968). Clinical signs of toxicity were diarrhoea, vomiting and haemorrhages in lung and
gastrointestinal tract. Microscopic analyses gave evidene of haemorrhage in many organs, particularly
the zona fasiculata of the adrenal glands and showed degenerative changes in the liver.
No data were identified for other farm species or companion animals.
In conclusion, the studies investigating the effects of Alternaria toxins as individual compounds on farm
and companion animals are scarce and are not sufficient to establish a NOAEL for animal species and to
estimate the relative potency of these mycotoxins. A lowest-observed-adverse-effect level (LOAEL) of
1.25 mg TeA/kg b.w. per day can be derived from the study performed by Giambrone et al. (1978) on
chickens.
7.
Risk characterisation
7.1.
Human health risk assessment
The database concerning toxicological effects of Alternaria toxins in experimental animals and/or in
humans is currently too limited to be used as a basis for identification of reference points for different
toxicological effects. Experiments performed in rodents with purified Alternaria toxins indicate that the
acute toxicity is in the following order: ALT > TeA > AME and AOH. However, these data are not
suitable for the risk assessment of Alternaria toxins since the risk for public health related to these
toxins is not expected to result from acute exposures.
Short term toxicity studies were performed according to non-standard protocols and do not provide
results that are suitable for risk assessment purposes.
A small number of studies indicate that AOH and AME may cause developmental effects in mouse
and/or hamster after subcutaneous or intraperitoneal administration of these mycotoxins. Nevertheless,
based on the route of administration, these studies can not be used for dietary risk assessment purposes.
Furthermore fetotoxic effects were not observed after oral exposure. One study considered the potential
for interaction between Alternaria toxins, suggesting synergism in the fetotoxic effects of AOH and
AME after subcutaneous administration. However, the limited information available, preclude
establishment of a common mode of action.
In vitro data provide clear evidence of the genotoxicity of Alternaria toxins such as AME and AOH.
There are no data on in vivo genotoxicity, and no convincing data on the carcinogenicity of these
compounds.
Considering the fact that there are few or no relevant toxicity data on Alternaria toxins, and that the
chemical structure of several of them is known, the CONTAM Panel considered it appropriate to use the
threshold of toxicological concern (TTC) approach to assess the relative level of concern of these
mycotoxins (Kroes et al., 2004; EFSA 2011c). Because it is essential for the application of the TTC
approach to have suitably conservative exposure estimates, which take into account the high exposure
scenarios, the CONTAM Panel based the assessment on the mean and 95th percentile chronic dietary
exposure to AOH, AME, TeA and TEN for adult population using LB and UB. The database for other
toxins is too weak for the application of the TTC.
EFSA Journal 2011;9(10):2407
51
In working through the TTC decision tree, it is necessary to assess the potential for genotoxicity.
Regarding Alternaria toxins, there is experimental evidence for in vitro genotoxicity of AOH and AME
in bacteria and mammalian cells. For such compounds, the TTC decision tree asks if the estimated
exposure exceeds the value of 2.5 ng/kg b.w. per day (0.15 g/person per day). In the adult population,
the mean chronic dietary exposure to AOH across dietary surveys ranged from 1.9 to 39 ng/kg b.w. per
day (range represents the minimum LB to maximum UB from the different countries). The
95th percentile dietary exposure ranged from 5.9 to 82 ng/kg b.w. per day. These values exceed the TTC,
indicating the need for additional toxicity data to assess the potential health risk. Although the exposure
estimates for AME were lower compared to those obtained for AOH (mean chronic dietary exposure
ranged from 0.8 to 4.7 ng/kg b.w. per day; 95th percentile dietary exposure ranged from 3.1 to 15 ng/kg
b.w. per day), the values for high consumers as well as the UB values for average consumers also
exceed the TTC, indicating a need for additional compound specific toxicity data.
Regarding TeA and TEN, for which there is no evidence of genotoxicity in bacteria or clear structural
alerts11 that raise concern for potential genotoxicity, the level defined by the TTC decision tree is
1500 ng/kg b.w. per day (90 g/person per day) for compounds in Cramer structural class III (Kroes et
al., 2004). For TEN, the mean chronic dietary exposure ranged from 36 to 141 ng/kg b.w. per day and
the 95th percentile dietary exposure ranged from 86 to 362 ng/kg b.w. per day, indicating that TEN is
unlikely to be of a human health concern. Estimates of chronic dietary exposure to TeA ( 13 ng/kg
b.w. per day) are much lower than the TTC value and TeA is therefore considered unlikely to be a
human health concern.
7.2.
Animal health risk assessment
At present, the knowledge on the possible effects of Alternaria toxins on farm and companion animals
as well as the database describing the occurrence of these contaminants in feedstuffs are weak and are
not sufficient to assess properly the risk regarding Alternaria toxins for animal health.
The estimation of the intake of AOH by chickens on the basis of UB values was found to be 6 g/day.
Since no evidence of toxicity was observed in chicks exposed during 3 weeks to levels approximately
17-fold higher than this value, it is unlikely that AOH represents a health risk for broilers or laying hens
at the present estimated level of intake.
Regarding TeA, the mean UB concentration in wheat as calculated from data reported in the literature is
990 g/kg. Unfortunately, the lack of data on other feed ingredients, namely maize and soybean meal,
does not allow an accurate calculation of TeA intake. Nevertheless, assuming that chicken could be fed
exclusively with wheat would result in a TeA intake of about 120 g/day, corresponding to
approximately 5 % of the LOAEL. This estimation suggests that adverse health effects of feed
containing TeA cannot be fully ruled out in chickens.
8.
Uncertainty analysis
The evaluation of the inherent uncertainties in the assessment of exposure to Alternaria toxins has been
performed following the guidance of the Opinion of the Scientific Committee related to Uncertainties in
Dietary Exposure Assessment (EFSA, 2006). In addition, the report on Characterizing and
Communicating Uncertainty in Exposure Assessment has been considered (WHO/IPCS, 2008).
According to the guidance provided by the EFSA opinion (2006) the following sources of uncertainties
have been considered: assessment objectives, exposure scenario, exposure model, and model input
(parameters).
11
Structural
alerts
were
analysed
by
using
as
software
tools
Toxtree
(v2.5)
(http://ihcp.jrc.ec.europa.eu/our_labs/computational_toxicology/qsar_tools/toxtree) and OECD QSAR Toolbox (v.2.1)
(http://www.oecd.org/document/54/0,3746,en_2649_34379_42923638_1_1_1_1,00.html)
EFSA Journal 2011;9(10):2407
52
8.1.
Assessment objectives
The objectives of the assessment for public and animal health were clearly specified in the terms of
reference. The CONTAM Panel assessed the occurrence data in food and feed that were collected from
the literature and EU MSs and carried out an exposure assessment for humans and animals. The
uncertainty in the assessment objectives is considered to be negligible.
8.2.
Exposure scenario and exposure model
The occurrence data in food provided were mainly from Germany and the data in feed were only from
the available literature. These data may not be representative of all food and feed commodities in which
Alternaria toxins could be present. This may have introduced uncertainties in the overall human and
animal exposure estimates. The high proportion of samples having levels < LOD or LOQ also has
introduced uncertainties to these estimates. The use of the UB approach for high percentage of
occurrence data < LOD and LOQ is conservative. The feed occurrence data may be overestimated due
to the fact that weather damaged samples were included in the calculations.
8.3.
Model input (parameters)
Only a limited number of Alternaria toxins have been identified and characterised. There are no
prescribed official methods or defined performance criteria for the analysis of Alternaria toxins and
laboratories can use any method of analysis. This may have added to the uncertainty in the analytical
results. Certified calibration standard solutions are available for AME, AOH and TEN analysis but not
for other Alternaria toxins which introduce uncertainties. The lack of certified reference materials and
proficiency tests are limitations and add thereby to the overall uncertainty.
Some evidence exists that Alternaria toxins have synergistic effects. They also could have combined
effects with other mycotoxins. The potential impact of such combined exposures cannot be predicted.
8.4.
Other uncertainties
There are major uncertainties due to the limited and inadequate toxicokinetics and toxicity data in
laboratory as well as in farm and companion animals.
8.5.
Summary of uncertainties
In Table 23, a summary of the uncertainty evaluation is presented, highlighting the main sources of
uncertainty and indicating an estimate of whether the respective source of uncertainty might have led to
an over- or underestimation of the exposure or the resulting risk.
EFSA Journal 2011;9(10):2407
53
Table 23: Summary of qualitative evaluation of the impact of uncertainties on the risk assessment of
the human and animal dietary exposure of Alternaria toxins.
Sources of uncertainty
Limited number of Alternaria toxins have been characterised
Lack of certified reference materials and proficiency tests
Occurrence data not representative of all food and feed commodities in which Alternaria toxins
could be present
Occurrence data not equally geographically distributed across Europe
Use of LB bound occurrence data in the dietary exposure estimations
Use of UB bound occurrence data in the dietary exposure estimations
Limited and inadequate toxicokinetic and toxicity data
Lack of carry over data
Direction
-(a)
+/+/+/+
+/-
LB: lower bound; UB upper bound; TTC: threshold of toxicological concern; (a): + = uncertainty with potential to cause overestimation of exposure/risk; - = uncertainty with potential to cause under-estimation of exposure/risk.
The CONTAM Panel considered the impact of the uncertainties on the risk assessment of animal and
human exposure to Alternaria toxins and concluded that overall uncertainty is large.
CONCLUSIONS AND RECOMMENDATIONS

CONCLUSIONS
General
Alternaria toxins are mycotoxins produced by Alternaria species. These fungi cause serious
diseases in many crops such as cereals, oil seeds, tomato and citrus fruits, and are also common
on dead organic matter.
Alternaria species are widespread in both humid and semi-arid regions and can infect growing
plants in the field, but also after harvest during storage and processing.
More than 70 Alternaria toxins have been reported and only some of them have been physicochemically characterised.
Alternaria toxins belong to 5 different classes (1) dibenzo--pyrones which include alternariol
(AOH), alternariol monomethyl ether (AME) and altenuene (ALT); (2) tenuazonic acid (TeA);
(3) perylene quinones which include altertoxins (ATX); (4) Alternaria alternata f. sp.
lycopersici toxins (AAL-toxins) and (5) miscellaneous structures such as tentoxin (TEN).
Methods of analysis
Several chromatography based techniques are suitable for Alternaria toxin quantification in
foods and feeds. Liquid chromatography coupled to (tandem) mass spectrometry has become
the method of choice due to its sensitivity, selectivity and specificity.
Reference standards are available for only some of the Alternaria toxins.
EFSA Journal 2011;9(10):2407
54
Occurrence and effect of processing
A total of 11730 data was considered in the assessment of Alternaria toxins in food: AOH
(n = 2291), AME (n = 2215), ALT (n = 1747), ATX-I (n = 1279), TeA (n = 1947), TEN
(n = 1388) and sum of AAL-toxins (n = 863). These include the data reported by two Member
States (84 %) and literature data reported for Europe (16%).
The reported data on Alternaria toxins in food were characterised by a high proportion of
results below the limit of detection (LOD)/limit of quantification (LOQ) ranging from 87 % up
to 100 % for the different compounds.
In samples containing Alternaria toxins, AOH, AME, TeA and TEN were generally found in
certain grains and grain-based products, tomato and tomato products, sunflower seeds and
sunflower oil, fruits and fruit products including fruit juices, and in beer and wine.
The highest concentrations for AOH, AME, TeA and TEN were found in the food group
Legumes, nuts and oilseeds and in particular in sunflower seeds. Mean concentrations of AOH
in this food group ranged from 22 g/kg (lower bound (LB) mean) to 26 g/kg (upper bound
(UB) mean) with a maximum value of 1200 g/kg. For AME the mean values were in the range
of 11 (LB) to 12 g/kg (UB), with a maximum value of 440 g/kg. TeA was present in higher
concentrations (LB mean = 333 g/kg; UB mean = 349 g/kg; maximum = 5400 g/kg). Mean
concentrations of TEN were in the range of 47 (LB mean) to 50 g/kg (UB mean) with a
maximum value of 880 g/kg.
No occurrence data on Alternaria toxins in feed were reported to the European Food Safety
Authority (EFSA).
According to published occurrence data on about 300 feed and agricultural commodities in
Europe, AOH was found in 31 % of the samples at concentrations from 6.3 to 1840 g/kg
(maximum found in sunflower seeds). AME was found in 6 % of the samples with levels
ranging from 3 to 184 g/kg (maximum found in cereals). ALT was found in 73 % of the
samples with concentrations between 6.3 and 41 g/kg (maximum found in wheat grains). TeA
was present in 15 % of the samples with levels varying between 500 and 4310 g/kg (maximum
found in oats).
Based on studies on occurrence of Alternaria toxins in feed worldwide, a total of 1150 results
on AOH (n = 755), AME (n = 158), ALT (n = 129) and TeA (n = 108) in feed were used in the
evaluation.
The results on Alternaria toxins in feed were characterised by a high proportion of results below
LOD/LOQ ranging from 9 % up to 66 % for the different compounds.
Scarce information is available on the behaviour of Alternaria toxins in food and feed during
the storage and processing. There are some indications that Alternaria toxin concentrations may
increase under favourable conditions and may be stable during food and feed processing.
Human exposure
Considering the limited occurrence data available and the high proportion of data below
LOD/LOQ (left-censored data), the Panel on Contaminants in the food chain (CONTAM Panel)
decided to perform a limited dietary exposure assessment, focussing only on adults ( 18 to
< 5 years old). This exposure estimate should be regarded as being only indicative.
EFSA Journal 2011;9(10):2407
55
Although the chronic dietary exposure was not calculated for all age classes, due to the higher
food consumption per kg body weight it is expected that the dietary exposure in children might
be higher compared to adults by a factor of 2 to 3. Similarly, vegetarians might have higher
exposure due to the higher intake of food of plant origin.
The dietary exposure was estimated only for AOH, AME, TeA and TEN where the quantified
results accounted for 7.7 %, 7.0 %, 13 % and 6.0 % of data, respectively. Due to the absence or
very limited number of quantified results for ALT (0 %), ATX-I (0.2 %) and AAL-toxins (0 %),
no dietary exposure assessment was performed for these toxins.
The estimated mean chronic dietary exposure in the adult population across dietary surveys,
using lower bound and upper bound concentrations, was in the following ranges: AOH: 1.9 39 ng/kg b.w. per day; AME: 0.8 - 4.7 ng/kg b.w. per day; TeA: 36 - 141 ng/kg b.w. per day;
TEN 0.01 - 7 ng/kg b.w. per day. The 95th percentile exposure estimates were 2 to 3 times
higher compared to the mean dietary exposure estimates.
Depending on the Alternaria toxins and the food consumption pattern in the European
countries, based on the few available data, the contribution to the dietary exposure to AOH,
AME, TeA and TEN is mainly made by grain and grain-based products, vegetables and
vegetable products in particular tomato products, fruits and fruit products including fruit and
vegetable juices, alcoholic beverages (wine and beer), oilseeds and vegetable oils (mainly
sunflower seeds and sunflower oil).
Animal exposure
Estimation of intake of Alternaria toxins in feed by farm livestock was limited to chicken, the
only species for which some toxicity data suitable for risk assessment of these toxins exist.
The occurrence data on feed were insufficient for most of the Alternaria toxins. Accordingly,
the estimates of exposure were limited to AOH. The calculated lower bound and upper bound
exposure to AOH is about 3 g/day and about 6 g/day, respectively, for both broilers and
laying hens. The exposure estimate should be regarded as being only indicative.
Hazard identification and characterisation

Toxicokinetics
No relevant information is available on the absorption, distribution and excretion of any

Alternaria toxin in animals and humans.
With the exception of AOH, AME and ALT, no data exist on the metabolism of Alternaria
toxins in mammals.
Available in vitro data indicate that AOH, AME and ALT are hydroxylated, mostly to catechol
metabolites, and form glucuronide and sulphate conjugates.
Toxicity in vitro and in vivo
AOH and AME are genotoxic in bacteria and mammalian cells in vitro. ATXs are mutagenic in
bacteria and induce cell transformation, while TEN and TeA are not mutagenic in bacteria.
Acute oral toxicity of TeA has been studied in several species (LD50 37.5 and 225 mg/kg b.w.).
EFSA Journal 2011;9(10):2407
56
Reproductive and developmental studies are limited. No data on toxic effects from oral
administration of Alternaria toxins have been reported.
There are no in vivo genotoxicity or carcinogenicity data available for Alternaria toxins. Some
indications of precancerous changes have been reported in oesophageal mucosa of mice.
Adverse effects in farm and companion animals
Data on sensitivity of farm and companion animals towards Alternaria toxins are very limited
and do not allow the estimation of tolerance levels for individual toxins and mixtures thereof.
Human risk characterisation
There are no relevant toxicity data for Alternaria toxins, for identification of reference points
for different toxicological effects.
The CONTAM Panel considered that the occurrence data concerning some of the Alternaria
toxins (AOH, AME, TeA and TEN) were adequate to apply the threshold of toxicological
concern (TTC) approach.
The estimated chronic dietary exposure to AOH and AME exceeded the relevant TTC value
indicating a need for additional compound-specific toxicity data.
The estimated chronic dietary exposures to TeA and TEN are lower than the relevant TTC value
and are therefore considered unlikely to be a human health concern.
Animal risk characterisation
It is unlikely that AOH represents a health risk for chicken but it can not fully be excluded that
TeA could be of concern for this species.
The lack of toxicological data precludes any conclusions for other species.
RECOMMENDATIONS
There is a need for certified reference materials and defined performance criteria for the
analysis of Alternaria toxins in various foods and feeds.
Representative occurrence data on Alternaria toxins in food and feed across the European
countries are required to refine exposure assessment.
More studies on the influence of food and feed processing on Alternaria toxins are needed.
More information is needed on the toxicokinetics, including metabolism, of the most

toxicologically relevant Alternaria toxins.
Toxicity data for AOH and AME are needed to enable their risk assessment.
Additional genotoxicity data for most of Alternaria toxins are required.
Information on susceptibility of farm and companion animals to Alternaria toxins is needed.
EFSA Journal 2011;9(10):2407
57
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EFSA Journal 2011;9(10):2407
69
APPENDICES
A. ALTERNARIA TOXINS PRODUCED BY ALTERNARIA SPP. AND PLANT DISEASES CAUSED BY
ALTERNARIA SPP.
The Alternaria toxins produced by Alternaria spp. and plant diseases caused by Alternaria spp. are
listed in Tables A1 and A2.
EFSA Journal 2011;9(10):2407
70
Table A1: Plant diseases caused by Alternaria spp.

Host
Citrus fruits
Lemons, Grapefruit, Mandarins and
Oranges (Citrus spp.)
Mandarins (C. reticulata Blanco)
Mexican lime (C. aurantiifolia (L.) Swingle)
Rough lemon (C. jambhiri Lush.) and
Rangpur lime (C. limonia Osbeck)
Pome fruits
Apple (Malus domestica Borkh.)
Disease(a)
Black rot/stem-end rot
Alternaria brown spot
Mancha foliar de
citricos
Alternaria leaf spot
rough lemon
Alternaria blotch
Alternaria rot
Nashi pear (Pyrus pyrifolia (Burm.) Nakai)
Black spot
Pear (P. communis L.)
Alternaria fruit rot
Stone fruits
Apricots, Plums, Sweet cherry, Peaches (Prunus
spp.)
Berries and small fruits
Grapes (Vitis spp.)
Strawberries (Fragaria ananassa Duch.)
Oilfruits
Olives (Olea europaea L.)
EFSA Journal 2011;9(10):2407
Causal organisms
Alternaria alternata (Fries) Keissl.
A. citri Ellis & N. Pierce
A. alternata (tangerine pathotype)
los A. limicola Simmons & Palm
of A. alternata (rough lemon pathotype)
A. alternata (apple pathotype)
References
Snowdon (1990a); Timmer et al. (2000)
Timmer et al. (2000)
Jones and
(1990a)
A. alternata
Jones and
A. tenuissima (Nees & Nees: Fries) Wiltshire (1990a)
A. alternata (Japanese pear pathotype)
Jones and
(1990a)
Alternaria spp.
Jones and
(1990a)
Aldwinckle (1990); Snowdon

Alternaria rot
A. alternata
Snowdon (1990a); Ogawa et al. (1995)
Berry and bunch rot
A. alternata
Alternaria fruit rot

Black leaf spot
A. alternata
A. tenuissima
A. alternata f. sp. fragariae Dingley
Pearson and Goheen (1988); Snowdon

(1990a)
Snowdon (1990a); Maas (1998)
Maas (1998)
Black rot
A. alternata
71
Table A1: Continued.

Host
Miscellaneous fruits
Avocados (Persea americana Mill.)
Figs (Ficus carica L.)
Mangoes (Mangifera indica L.)
Papaya (Carica papaya L.)
Passion fruit (Passiflora edulis Sims)
Persimmon (Diospyros kaki L.f.)
Tree nuts
Hazelnut (Corylus avellana L.)
Pistachio (Pistacia vera L.)
Walnut (Juglans regia L.)
Legumes
Beans (Phaseolus vulgaris L.)
Chick pea (Cicer arietinum L.)

Peanut (Arachis hypogaea L.)
Peas (Pisum sativum L.)
Soya beans (Glycine max (L.) Merrill)
Grains
Barley (Hordeum vulgare L.)
Maize (Zea mays L.)
Rice (Oryza sativa L.)
Rye (Secale cereale L.)
Wheat (Triticum spp. L.)
EFSA Journal 2011;9(10):2407
Disease(a)
Causal organisms
References
Alternaria rot
Fungal decay
Alternaria black spot
Alternaria fruit spot
Brown spot
Snowdon (1990a)
Doster and Michaillides (2007)
Snowdon (1990a); Ploetz et al. (1994)
Snowdon (1990a); Ploetz et al. (1994)
Snowdon (1990a)
Alternaria rot
A. alternata
A. alternata
A. alternata
A. alternata
A. alternata
A. passiflorae J.H. Simmonds
A. alternata
Brown apical necrosis

Alternaria late blight
Grey necrosis
A. alternata
A. alternata
A. alternata
Hong et al. (2006); Andrew et al. (2009)

Teviotdale et al. (2002)
Hong et al. (2006); Andrew et al. (2009)
Snowdon (1990a)
Alternaria leaf and pod A. alternata

spot
A. brassicae (Berk.) Sacc. f. sp. phaseoli
Brun.
A. brassicicola (Schw.) Wiltshire
A. brasiliensis Queiroz, Muniz & Menezes
Alternaria blight
A. alternata
Alternaria leaf blight
A. alternata
A. arachidis R.L. Kulk.
Alternaria blight
A. alternata
Alternaria leaf and pod A. tenuissima
necrosis
Hall (1991); Queiroz et al. (2000)
Kernel blight
Ear rot
Stackburn
Storage molds
Mathre (1997)
White (1999)
Webster and Gunnell (1992)

Black head molds
Alternaria spp.
A. alternata
A. padwickii (Ganguly) M.B. Ellis
A. alternata
Alternaria spp.
A. triticina Prasada & Prabhu
A. alternata,Alternaria spp.
Chen et al. (2011)

Porter et al. (1990)
Hagedorn (1984)
Hartman et al. (1999)
Wiese (1987).
Wiese (1987).
72

Host
Oilseeds
Cotton (Gossypium spp.)
Linseed (Linum spp.)
Mustard (Brassica juncea (L.) Czern. and B.
nigra (L.) W.D.J.Koch)
Rape seed (B. napus L. and B. rapa L.)
Safflower (Carthamus tinctorius L.)

Sesame (Sesamum indicum L.)
Sunflower (Helianthus annuus L.)
Root vegetables
Beetroot (Beta vulgaris L.)
Carrots (Daucus carota L.)
Radishes (Raphanus sativus L.)
Bulb vegetables
Garlic (Allium sativum L.)
Onions (A. cepa L.)
EFSA Journal 2011;9(10):2407
Disease(a)
Causal organisms
Leaf spot
A. macrospora Zimm.
A. linicola J.W. Groves & Skolko
Alternaria black spot
A. brassicae
A. brassicicola
A. japonica Yoshii
Dark pod spot
A. brassicae
A. brassicicola
A. japonica
A. carthami Chowdhury
A. alternata
A. sesami (Kawam.) Mohanty & Behera
A. sesamicola Kawam.
Alternaria leaf blight, stem A. alternata
spot and head rot
A. helianthi (Hansf.) Simmons
A. tenuissima
A. zinniae M.B. Ellis
A. alternata
A. brassicae
A. dauci (Khn) J.W. Groves & Skolko
Black rot (black carrot root A. radicina Meier, Drechsler and Eddy
dieback)
Black spot
A. brassicae
A. brassicicola
A. japonica
Purple blotch
A. porri (Ellis) Ciferri
References
Kirkpatrick and Rothrock (2001)
Vloutoglou et al. (1999)
Rimmer et al. (2007)
Irwin (1976)
Yu et al. (1982)
McDonald and Martens (1963); Allen et
al. (1981); Narain and Chauhan (1981);
Lagopodi and Thanassoulopoulos (1998)
Harveson et al. (2009)
Davis and Raid (2002)
Davis and Raid (2002)
Schwartz and Mohan (2008)
73

Host
Stem vegetables
Fennel (Foeniculum vulgare Mill.)
Leek (A. ampeloprasum L.)
Fruiting vegetables
Aubergines (Solanum melongena L.) and
Peppers (Capsicum annuum L.)
Cucumbers (Cucumis sativus L.),
Melons (C. melo L.),
Pumpkins (Cucurbita maxima Duchesne) and
Watermelons (Citrullus lanatus (Thunb.) Mats.
& Nakai)
Tomatoes (Lycopersicum esculentum Mill)
Brassica vegetables
Broccoli, Cauliflowers, and
Cabbage (B. oleracea L.)
Leaf vegetables
Endive (Cichorium endivia L.)
Tea and herbs for infusions
Tea (Camellia sinensis (L.) Kuntze)
Potatoes (Solanum tuberosum L.)
Sweet potates (Ipomoea batatas (L.) Lam.)
Miscellaneous crops
Hop (Humulus lupulus L.)
Tobacco (Nicotiana tabacum L.)
Disease(a)
Causal organisms
References
Leaf spot
Purple blotch
A. petroselini (Neerg.) E.G. Simmons

A. porri
Infantino et al. (2009)

Schwartz and Mohan (2008)
Alternaria rot
A. alternata
Snowdon (1990b); Pernezny et al. (2003)

Alternaria rot
A. cucumerina (Ellis & Everh.) J.A. Elliott

A. alternata f. sp. cucurbitae Vakal.
A. alternata
A. cucumerina
Zitter et al. (1996)

Zitter et al. (1996)
Snowdon (1990b); Zitter et al. (1996)
Alternaria stem canker

Black mold rot
Early blight
A. alternata f. sp. lycopersici Keissl.

A. alternata
A. solani Sorauer
Jones et al. (1991)

Jones et al. (1991)
Jones et al. (1991)
Black spot
A. brassicae
A. brassicicola
A. japonica
Leaf spot
A. cichorii Nattrass
Barreto et al. (2008)
Alternaria blight
A. alternata
Chakraborty et al. (2006)
Brown spot and black pit

Early blight
Alternaria leaf spot, stem
blight and storage rot
A. alternata
A. solani
A. alternata
Alternaria spp.
Stevenson et al. (2001)

Stevenson et al. (2001)
Clark and Moyer (1988)
Alternaria blight
Alternaria cone disorder
Brown spot
A. humuli Simmons
A. alternata
A. alternata
Simmons (2007)
Mahaffee et al. (2009)
Shew and Lucas (1991)
(a): According to the Committee on Standardization of Common Names for Plant Diseases, American Phytopathological Society.
EFSA Journal 2011;9(10):2407
74
Table A2: Toxins produced by Alternaria spp.

Species(a)
Alternaria alternata (Fries) Keissl.
Dibenzo-pyrones
AOH
AME
ALT
Altenuisol
Altenusin
Dehydroaltenusin
Isoaltenuene
5'-Epialtenuene
Neoaltenuene
Toxins(b)
Tenuazonic acid
Perylene quinones
TeA
ATX-I
ATX-II
ATX-III
Alterlosin
Stemphyltoxin
References
Other major metabolites
AS-toxin
Altenuic acids
TEN
Maculosin
Alterlosins
Dihydrotentoxin
Montemurro and Visconti (1992);

Liakopoulou-Kyriakides
et
al.
(1997); Bottalico and Logrieco
(1998); Thomma (2003)
A. alternata f. sp. fragariae Dingley
AF-toxin
A. alternata f. sp. lycopersici Keissl.
AAL-toxins
TEN
ACTG-toxin
Montemurro and
Thomma (2003)
Montemurro and
Thomma (2003)
Montemurro and
Thomma (2003)
Montemurro and
Thomma (2003)
Altenin
Dihydroisocoumarin
AK-toxin

Bottalico and Logrieco (1998);
Thomma (2003)
Alternaric acid
TEN
AM-toxin
Macrosporin
Methylanthraquinone

Thomma (2003)
Bottalico and Logrieco (1998)
A. alternata (rough lemon pathotype)
TeA
ACR-toxin
A. alternata (tangerine pathotype)

A. alternata (Japanese pear
pathotype)
(= A. gaisen Nagano)
A. alternata (apple pathotype)
(= A. mali Roberts)
A. bataticola Ikata
EFSA Journal 2011;9(10):2407
AOH
AME
Altenusin
Dehydroaltenusin
TeA
TeA
ATX-I
ATX-II
ATX-III
Visconti (1992);
Visconti (1992);
Visconti (1992);
Visconti (1992);
75

Species(a)
A. brassicae (Berk.) Sacc.
A. brassicicola (Schw.) Wiltshire
A. capsici-annui Svul. & Sandu
Dibenzo--pyrones
AOH
AME
AOH
AME
AOH
AME
ALT
Toxins(b)
Tenuazonic acid
Perylene quinones
TeA
TeA
A. cucumerina (Ellis & Everh.) J.A.

Elliott
EFSA Journal 2011;9(10):2407
ATX-I
AOH
AME
AOH
AME
ALT
AOH
AME

Thomma (2003)
Zinniol
Brefeldin
Dehydrobrefeldin
TeA
A. chrysanthemi E.G. Simmons &

Crosier (curr. name Teretispora
leucanthemi (Nelen) E.G.
Simmons)
A. cichorii Nattrass
A. cinerariae Hori & Enjoji
A. citri Ellis & N. Pierce

Destruxin
Homodestruxin
Desmethyldestruxin
TeA
A. carthami Chowdhury
A. cheiranthi (Lib. : Fr.) P.C. Bolle
References

Radicinin
Radicinol

Zinniols
Curvularin
Dehydrocurvularin

Montemurro and Visconti (1992)
TeA

Curvularin
Macrosporin
Methylanthraquinone

76

Species(a)
A. dauci (Khn) J.W. Groves &
Skolko
Dibenzo--pyrones
AOH
AME
Toxins(b)
Tenuazonic acid
Perylene quinones
A. eichorniae Nag Raj & Ponnappa

A. helianthi (Hansf.) Tubaki &
Nishih. (curr. name Alternariaster
helianthi (Hansf.) E.G. Simmons)
A. infectoria (E.G. Simmons)
(current name Lewia infectoria
(Fuckel) M.E. Barr & E.G.
Simmons)
A. japonica Yoshii
A. longipes (Ellis & Everh.) E.W.
Mason
A. macrospora Zimm.
A. porri (Ellis) Ciferri
A. oryzae Hara
EFSA Journal 2011;9(10):2407
Alteichin
References
Zinniol
Bostrycin
Deoxybostrycin
Deoxyradicinin
Pyrenocines

Montemurro and Visconti (1992)
AOH
AME
TeA
AOH
AME
AME
TeA
TeA

AME
ALT
TeA
Zinniol
Altersolanols
TEN
Porritoxin
Physcion
Porriolide
Zinniol
Macrosporin
Alterporriol
Methylanthraquinone
Alternaric acid

77

Species(a)
A. radicina Meier, Drechsler &
Eddy
A. solani Sorauer
A. tagetica S.K. Shome & Mustafee

A. tenuissima (Nees & Nees: Fries)
Wiltshire
A. tomato (Cooke) L.R. Jones
A. zinniae M.B. Ellis
Dibenzo--pyrones
ALT
Toxins(b)
Tenuazonic acid
Perylene quinones
TeA
ATX-I
AOH
AME
AOH
AME
ALT
AOH
AME
ALT
References
Radicinin
Radicinol
Alternaric acid
Altersolanols
Tetrahydroaltersolanol
Alterporriols
Solanapyrones
Zinniol
Zinnolide
Macrosporin
Zinniol
TeA
ATX-I
TeA
ATX-I
ATX-II
Curvularin
Dehydrocurvularin
Hydroxycurvularin
Zinniol


(a): Source: Index Fungorum CAB International (http://www.indexfungorum.org); Simmons (2007); Andrew et al. (2009); (b): AOH: alternariol; AME: alternariol monomethyl ether; ALT:
altenuene; TeA: tenuazonic acid; ATX: altertoxin; TEN: tentoxin; AS-toxin: from A. alternata pathogenic to sunflower; AF-toxin: from A. alternata f. sp. fragariae; AAL-toxins: from A.
alternata f. sp. lycopersici; ACR-toxin: from A. alternata rough lemon pathotype, former A. citri; ACTG-toxin: from A. alternata tangerine pathotype, former A. citri; AK-toxin: from A.
alternata Japanese pear pathotype, former A. kikuchiana S. Tanaka; AM-toxin: from A. mali.
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78
B. THE OCCURRENCE DATA ON FOODS AND FEEDS REPORTED IN THE LITERATURE

The occurrence data on foods and feeds reported in the literature are presented in the Table B1 and Table B2, respectively.
Table B1: Occurrence of Alternaria toxins in food (in g/kg or g/L(a)) reported in the literature.
Region/Country
Toxin
Commodity
n > LOQ
LOD(b)/
LOQ(c)
Median(d)
AOH
AME
ALT
TeA
Lentils
Lentils
Lentils
Lentils
Peas, green, without pods
Oilseeds (linseed and fibre flax
seed)
seed)
seed)
3
3
3
3
3
0
0
0
15
15
75
84
122
Mean
Maximum
Method
Reference
290
n.d.
n.d.
n.d.
HPTLC
Ostry et al. (2004)(e)
n.d.
104
(c)
n.d.
(c)
30
HPLC-FLD
Krlov et al. (2006)
HPTLC
Ostry et al. (2007)
Europe
Czech Republic
AOH
Czech Republic
AME
ALT
AOH
Wine
Must
Grape juice
AME
Wine
Must
Grape juice
ALT
Wine
Must
Grape juice
TeA
Wine
Must
Grape juice
Czech Republic
EFSA Journal 2011;9(10):2407
84
122
20
84
n.d.
122
13
13
13
13
13
13
13
13
13
13
13
13
2
0
0
0
0
0
0
0
0
0
0
0
0
9
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.5
1.5(a)(c)
1.5
7.5
79
Table B1: Continued.

Region/Country
Toxin
Germany
AOH
Germany
TEA
AOH
Germany
AME
EFSA Journal 2011;9(10):2407
Commodity
Apple sauce
Apple juice
Tomato ketchup
Tomato paste
Tomato juice
White wine
Cereals
Cereals
Red/white wine
Red grap juice
Carrot juice
Vegetable juice
Tomato puree
Tomato ketchup
Steack sauce
Tomato paste
Tomato juice
Cereals
Red/white wine
Red grap juice
Carrot juice
Vegetable juice
Tomato puree
Tomato ketchup
Steack sauce
Tomato paste
Tomato juice
N
10
44
18
10
16
11
27
13
6
1
1
2
3
1
1
1
1
13
6
1
1
2
3
1
1
1
1
n > LOQ
(e)
6
9(e)
18(e)
10(e)
9(e)
2(e)
2(e)
1
4
0
0
0
2
1
0
1
1
1
4
0
0
1
2
1
0
1
1
LOD(b)/
LOQ(c)
1(b)
0.059(a)
1
2
0.059(a)
0.059(a)
50(c)
1.5
0.1
0.1
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3(c)
0.03
0.03
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Mean
Median(d)
(d)
1.5 / 1.4
2.4 /2.2(a)
2.5 / 2.2
6.6 / 5.7
1.9 / 1.6(a)
1.9 / 1.6(a)
49
-
Maximum
2
3.5(a)
5.0
13
3.1(a)
2.0(a)
851
4.1
4.9
n.d.
n.d.
n.d.
5.9
2.6
n.d.
25
5.4
0.6
0.3
n.d.
n.d.
0.1
0.5
0.4
n.d.
5.3
0.9
Method
Reference
pAb EIA
Ackermann et al., 2011
HPLC-MS/MS
Siegel et al. (2009)(f)
HPLC-MS/MS
Asam et al. (2010)(f)
80

Region/Country
Germany
Toxin
TeA
AOH
Germany
AME
Germany
AOH
AME
ALT
ATX-I
EFSA Journal 2011;9(10):2407
Commodity
Bottled Beers
White wine
Red wine
Mulled wine
Fruit punch
Grape juice
Apple juice
Orange juice
Tomato juice
Vegetable juice
White wine
Red wine
Mulled wine
Fruit punch
Grape juice
Apple juice
Orange juice
Tomato juice
Vegetable juice
Fruits and Vegetables
Fruits and vegetables(g)
n > LOQ
43
6
5
2
1
5
3
2
2
1
6
5
2
1
5
3
2
2
1
47
47
47
47
16
6
5
2
1
5
3
2
2
1
0
4
0
1
0
0
2
2
1
0
0
0
0
LOD(b)/
LOQ(c)
8(c)
0.09
0.09
0.09
0.09
0.09
0.09
0.09
0.09
0.09
0.03(c)
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
10-400
25(b)-350
40-800
70-1000
Mean
Median(d)
11
1.13
4.50
0.11
0.17
0.20
1.26
0.08(d)
0.23
0.31
-
Maximum
Method
Reference
175
7.59
7.50
2.70
0.27
1.05
0.22
0.24
1.99
7.82
n.q.
0.15
n.q.
0.04
n.q.
n.q.
0.27
0.38
0.79
n.d.
n.d.
n.d.
n.d.
LC-ESI-IT MS
Siegel et al. (2010b)(f)
HPLC-MS/MS
Asam et al. (2009)
HPLC-UV
Wittkowski et al. (1983)
81

Region/Country
AOH
AME
Italy
ALT
ATX-I
TeA
Italy
n > LOQ
Olives
Olive husks
Olive oil
Olives
Olives husks
Olive oil
Olives
Olive husks
Olive oil
Olives
Olive husks
Olive oil
Olives
Olive husks
Olive oil
9
3
6
9
3
6
9
3
6
9
3
6
9
3
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
LOD(b)/
LOQ(c)
50
50
50
30(b)
30
30
100
100
100
200
200
200
100
100
100
Cornflakes
29(a)(c)
Toxin
TeA
Commodity
(h)
AOH
Poland
AME
EFSA Journal 2011;9(10):2407
Fruit and vegetables

Fruit juices
Fruit drinks
Tomato juices
Tomato concentrates
Fruit and vegetables(h)
Fruit juices
Fruit drinks
Tomato juices
Tomato concentrates
12
24
17
12
6
4
12
24
17
12
6
4
0
0
0
0
0
0
0
0
0
0
0
0
(b)
3
3
3
3
3
3
3(b)
3
3
3
3
3
Mean
Max
Method
Reference
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
HPTLC
Visconti et al. (1986)(f)
25
HPLCUV/DAD
Aresta et al. (2003)
n.d.
n.q.
n.d.
n.q.
n.q.
n.q.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
TLC-UV
Gyrin and Szteke

(1995)(f)
Median(d)
82

Region/Country
Spain
Spain
The Netherlands,
France, Denmark
Toxin
Commodity
n > LOQ
AOH
AME
AOH
AME
AOH
AME
ATX-I
TeA
Apple juice concentrates

Apple juice concentrates
Apple juices
Apple juices
Carrots (organic)
Carrots (organic)
Carrots (organic)
Carrots (organic)
32
32
7
7
266
266
266
266
16
1
0
1
0
0
0
0
TeA
Ice-wine
26
Red wines
White wines
Imported red wines
Imported white wines
Red grape juices
White grape juices
Cranberry juices
Red wines
White wines
Imported red wines
Imported white wines
Red grape juices
White grape juices
Cranberry juices
17
19
7
4
10
4
5
17
19
7
4
10
4
5
13
1
7
1
3
0
1
13
2
6
0
5
0
0
LOD(b)/
LOQ(c)
0.8
0.8(c)
1.6
0.7(b)
5
10(b)
20
20
Mean
Max
Method
Reference
5.42
1.71(a)
n.d.(a)
0.85
n.d.
n.d.
n.d.
n.d.
HPLCUV/DAD
Delgado and GomezCorvods (1998)
LC-UV
Delgado et al. (1996)
LC-UV
Solfrizzo et al. (2004)
70(b)
n.d.
LC-DAD
Abramson et al.
(2007)
0.01-0.8
0.01-0.8
0.01-0.8
0.01-0.8
0.01-0.4
0.01-0.4
0.01-0.4
0.01(b)-0.5
0.01-0.5
0.01-0.5
0.01-0.5
0.01-0.4
0.01-0.4
0.01-0.4
5.0
1.48
7.41
0.67
0.46
n.d.
0.04
0.23(a)
0.06
0.19
n.d.
39.5
n.d.
n.d.
LC-MS/MS
Scott et al. (2006)(f)
Median(d)
America
Canada
AOH
Canada
AME
EFSA Journal 2011;9(10):2407
83

Region/Country
Toxin
Commodity
n > LOQ
LOD(b)/
LOQ(c)
AOH
Apple juice
11
0.01 0.08
2.40
Other fruit beverages
0.3(b) -0.7
5.60
Canada
(b)
AME
Canada
Argentina
AOH
AME
AOH
AME
TeA
AOH
Brazil
(b)
Mean
Median(d)
AME
TeA
Max
Apple juice
11
0.01 0.08
0.43(a)
Other fruit beverages

Apple juices
Apple juices
Tomato puree
Tomato puree
Tomato puree
Tomato juice
Stewed tomato
Tomato paste
Tomato pulp
Tomato pure
Tomato juice
Stewed tomato
Tomato paste
Tomato pulp
Tomato pure
Tomato juice
Stewed tomato
Tomato paste
Tomato pulp
Tomato pure
9
8
8
80
80
80
11
1
24
22
22
11
1
24
22
22
11
1
24
22
22
3
3
0
5
21
23
0
0
0
0
0
0
0
0
0
0
0
0
0
7
4
0.1-0.5
1
1(b)
5
2(c)
11
5
5
5
5
5
2(b)
2
2
2
2
11
11
11
11
11
1.4
5.0
n.d.(a)
8756
1734
4021
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
111
76
Method
Reference
LC-MS/MS
Lau et al. (2003)(f)
GC-MS
Scott et al. (1997)(f)
LC-UV
Terminiello et al.
(2006)(f)
HPLC-DAD
da Motta and
Valente Soares
(2001)(f)
N: numer of samples; LOQ: limit of quantification; n > LOQ: number of samples > LOQ; LOD: limit of detection; Max: maximum; AOH: alternariol; AME: alternariol monomethyl ether;
ALT: altenuene; TeA: tenuazonic acid; ATX: altertoxin; n.d.: not detected; -: not available; n.q.: not quantified; (a): g/L; (b): LOD; (c): LOQ; (d): median; (e): n of samples > LOD;
f):Individual data available; (g): Fruit and vegetables = apples, apricots, raspberries, gooseberries, strawberries, blackcurrants and coloured currants, blueberries, cherries, tomatoes and carrots;
(h): not specified but LODs were given for some fruit juices (apple, pear, white grape, red currant, cherry, peach, orange, grapefruit, tomato, multi-fruit), jam (strawberry, cherry), plum mush,
tomato (ketchup, pure) and baby food.
EFSA Journal 2011;9(10):2407
84
Table B2: Occurrence data of Alternaria toxins in agricultural commodities and feed (in g/kg) reported in the literature.
Region/Country
Alternaria
toxin
n > LOD(a)/LOQ(b)/
Mean
LOQ CC(c)/CC(d)
Minimum
Maximum
12(c)/23(d)
n.d.
n.d.
n.d.
14
12/23
n.d.
17
Wheat grain
12/23
n.d.
n.d.
n.d.
Sow feed
18/39
n.d.
n.d.
n.d.
Maize grain
14
18/39
n.d.
19
Wheat grain
18/39
n.d.
n.d.
n.d.
Maize grain
12/23
19
25
Wheat grain
12/23
n.d.
n.d.
n.d.
Maize grain
18/39
n.d.
n.d.
n.d.
Wheat grain
18/39
n.d.
n.d.
n.d.
Wheat grain
129
60
2(a)/5(b)
7.7
6.3
44.4
2/5
n.d.
21
129
2/5
n.d.
n.d.
n.d.
2/5
n.d.
n.d.
n.d.
129
118
2/5
18.7
6.3
41
2/5
n.d.
30
129
10/25
n.d.
n.d.
n.d.
10/25
n.d.
n.d.
n.d.
Commodity
Sow feed
Maize grain
Method
Reference
LC-MS/MS
Monbaliu et al. (2010); S.

Monbaliu (2011), personal
communication.
LC-MS/MS

communication
HPTLC
Europe
AOH
Belgium
AME
AOH
Czech Republic
AME
AOH
AME
Czech Republic
ALT
TeA
EFSA Journal 2011;9(10):2407
Rape seed
Wheat grain
Rape seed
Wheat grain
Rape seed
Wheat grain
Rape seed
85

Region/Country
Alternaria
toxin
Commodity
AOH
Wheat grain
14
n > LOD(a)/LOQ(b)/
LOQ CC(c)/CC(d)
0
12(c)/23(d)
Mean
Minimum
Maximum
n.d.
n.d.
n.d.
Denmark
AME
AOH
Denmark
AME
Estonia
Germany
AOH
Wheat grain
14
18/39
n.d.
n.d.
n.d.
Maize silage
10
10(a)
24
n.d.
24
Maize silage
(samples with
visible fungal
growth)
10
10
236
n.d.
236
Maize silage
10
n.d.
n.d.
n.d.
Maize silage
(samples with
visible fungal
growth)
10
51
n.d.
51
Oats grain
100(b)
n.d.
n.d.
n.d.
Wheat grain
100
210
340
Barley grain
100
n.d.
130
(a)
13
250
100
(b)
AOH
Oat flour
0.5 /1.5
AME
Oat flour
0.1/0.3
EFSA Journal 2011;9(10):2407
Method
Reference
LC-MS/MS

communication
LC-MS/MS
Rasmussen et al. (2010)
HPLC-DAD
Ktt et al. (2010)
LC-MS/MS
Asam et al. (2010)
86

Region/Country
Hungary
Maximum
12(c)/23(d)
n.d.
n.d.
n.d.
18/39
n.d.
n.d.
n.d.
50(a)
1840
1840
AOH
Wheat grain
AME
Wheat grain
Italy
AME
Spain
Minimum
Commodity
AOH
Portugal
n > LOD(a)/LOQ(b)/
Mean
LOQ CC(c)/CC(d)
Alternaria
toxin
Sunflower
seed (visibly
affected by
Alternaria
rot)
Sunflower
seed (visibly
affected by
Alternaria
rot)
30
n.d.
129
AOH
Maize grain
12
12(c)/23(d)
n.d.
n.d.
n.d.
AME
Maize grain
12
18/39
n.d.
n.d.
n.d.
AOH
Maize grain
14
12/23
n.d.
n.d.
n.d.
AME
Maize grain
14
18/39
n.d.
n.d.
n.d.
EFSA Journal 2011;9(10):2407
Method
Reference
LC-MS/MS

communication
HPLC-UV
LC-MS/MS
LC-MS/MS

communication
communication
87

Region/Country
Alternaria
toxin
AOH
Sweden
AME
TeA
AOH
United Kingdom
AME
ALT
ATX-I
United Kingdom
TeA
Commodity
Barley, wheat,
oats, straw
Barley, wheat,
oats, straw
Barley, wheat,
oats, straw
Rape seed
meal
Rape seed
meal
Rape seed
meal
Rape seed
meal
Rape seed
meal
n > LOD(a)/LOQ(b)/
Mean
LOQ CC(c)/CC(d)
Minimum
Maximum
18
16
35(a)/45(b)
n.d.
335
18
35/45
n.d.
184
18
18
100/135
980
4310
30
50(b)
68
54
81
30
40
55
49
60
30
40
n.d.
n.d.
n.d.
30
200
n.d.
n.d.
n.d.
30
350
730
500
970
Method
Reference
HPLC-UV
Hggblom et al. (2007);

P. Hggblom (2011),
personal communication
HPLC-FLD
Nawaz et al. (1997)
HPLC-UV
Nawaz et al. (1997)
HPLC-MS
Mansfield et al. (2007)
direct competitive
ELISA
Yu et al. (1999)
North America
Pennsylvania
AAL-TA
Maize silage
120
28
20(a)
170
200
2000
AAL-TB
Maize silage
120
17
20
50
30
900
25
25
50(b)
720
290
1160
AAL-TA
Wisconsin
AAL-TA
EFSA Journal 2011;9(10):2407
Hay and hay

silage
Maize germ
meal, maize
silage and
other mixed
feed
38
36
50
90
1470
88

Region/Country
Alternaria
toxin
Commodity
n>
LOQ
LOD(a)/LOQ(b)/
CC(c)/CC(d)
Mean
Minimum
Maximum
AOH
Wheat grain
64
50(a)
1054
645
1388
AME
Wheat grain
64
15
50
2118
566
7451
TeA
Wheat grain
Sunflower
seed
Sunflower
seed
Sunflower
seed
Sunflower
seed
Sunflower
seed
64
12
80
2313
1001
8814
150
128
50(a)
187
50
676
150
70
30
194
30
836
150
98
200
6692
2500
15796
50
37
50(a)
35
792
50
31
30
90
630
Method
Reference
HPLC-UV
HPLC-UV
Chulze et al. (1995)
TLC
Chulze et al. (1995)
TLC
Torres et al. (1993)
HPLC-UV
Barros et al. (2011) ;

Ramirez (2011),
personal
communication;
South America
Argentina
AOH
Argentina
AME
Argentina
Argentina
TeA
AOH
AME
Argentina
AOH
Soya bean
15
15(e)
8(a)
72
25
141
AOH
Soya bean
35
8(e)
70
22
211
16
363
75
1153
16
277
62
483
8(a)
72
25
141
AME
Soya bean
15
15
(e)
(e)
AME
Soya bean
35
AOH
Soya bean
15
15(e)
EFSA Journal 2011;9(10):2407
89

Region/Country
Alternaria
toxin
LOD(a)/LOQ(b)/
Mean
CC(c)/CC(d)
Commodity
n>
LOQ
Maize grain
Wheat grain
Barley grain
Sorghum
Wheat bran
Rice
Poultry feed
Cotton seed
cake
Peanut oil cake
Sunflower seed
cake
Maize grain
15
15
10
10
10
7
20
0
4
0
0
2
0
0
50(a)
50
50
50
50
50
50
15
10
Minimum
Maximum
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2320
n.d.
n.d.
310
n.d.
n.d.
50
n.d.
n.d.
n.d.
50
n.d.
85
18
50
n.d.
n.d.
n.d.
15
300
n.d.
n.d.
n.d.
Wheat grain
15
300
n.d.
1890
Barley grain
10
300
n.d.
300
Sorghum
Wheat bran
Rice
Poultry feed
Cotton seed
cake
Peanut oil cake
Sunflower seed
cake
10
10
7
20
0
1
0
4
300
300
300
300
n.d.
n.d.
-
n.d.
n.d.
n.d.
n.d.
n.d.
460
n.d.
270
15
300
n.d.
n.d.
n.d.
10
300
n.d.
335
18
300
n.d.
125
Method
Reference
HPLC-UV
Abd el-Aal (1997)
Africa
Egypt
Egypt
AOH
AME
EFSA Journal 2011;9(10):2407
90

Region/Country
Alternaria
toxin
ALT
Egypt
ATX-I
EFSA Journal 2011;9(10):2407
LOD(a)/LOQ(b)/
Mean
CC(c)/CC(d)
100
100
100
100
n.d.
100
n.d.
100
100
100
100
-
Commodity
Maize grain
Wheat grain
Barley grain
Sorghum
Wheat bran
Rice
Poultry feed
Cotton seed cake
Peanut oil cake
Sunflower seed
cake
Maize grain
15
15
10
10
10
7
20
15
10
n>
LOQ
3
2
1
0
0
2
3
1
1
18
100
15
Wheat grain
Barley grain
Sorghum
15
10
10
Wheat bran
Rice
Poultry feed
Cotton seed cake
Peanut oil cake
Sunflower seed
cake
Minimum
Maximum
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
370
1480
700
n.d.
n.d.
100
140
800
650
n.d.
185
200
n.d.
n.d.
n.d.
2
0
2
200
200
200
n.d.
-
n.d.
n.d.
n.d.
1678
n.d.
185
10
7
20
0
0
3
200
200
200
n.d.
n.d.
-
n.d.
n.d.
n.d.
n.d.
n.d.
880
15
10
0
1
200
200
n.d.
-
n.d.
n.d.
n.d.
400
18
200
n.d.
n.d.
n.d.
Method
Reference
HPLC-UV
Abd el-Aal (1997)
91

Region/Country
Egypt
South Africa
N
15
15
10
10
10
7
20
15
100
10
18
AOH
Maize grain
Wheat grain
Barley grain
Sorghum
Wheat bran
Rice
Poultry feed
Cotton
seed
cake
Peanut oil cake
Sunflower
seed cake
Sorghumbased mixed
feed (swine)
Wheat grain
Barley grain
Maize grain
Wheat bran
Soya bean oil
meal
Sunflower
meal and oil
cake
Maize gluten
Combined
feeds
Hay
n>
LOQ
4
5
0
3
4
2
8
AOH
Silage
TeA
AME
AOH
AOH
AOH
AOH
AOH
Russia
LOD(a)/LOQ(b)/
Mean
CC(c)/CC(d)
100
100
100
n.d.
100
100
100
100
-
Commodity
Alternaria
toxin
AOH
AOH
AOH
EFSA Journal 2011;9(10):2407
Minimum
Maximum
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
253.6
658
n.d.
125
200.5
172.8
295.7
n.d.
346.6
100
n.d.
n.d.
n.d.
100
n.d.
n.d.
n.d.
10(a)
1837
1200
2250
28
76
52
10
4
22
5
4
20(b)
20
20
20
98
47
88
34
76
20
38
20
192
126
169
63
20
23
21
17
20
192
25
388
20
21
6
1
16
20
56
20
190
59
20
49
20
334
20
560
20
760
Method
Reference
HPLC-UV
Abd el-Aal (1997)
HPLC-FLD
Sydenham et al. (1988)
ELISA
Burkin and Kononenko

(2011)
92

Region/Country
Alternaria
toxin
Commodity
n>
LOQ
LOD(a)/LOQ(b)/
CC(c)/CC(d)
Mean
Minimum
Maximum
22
20
50(a)
335
116
731
22
21
50
443
52
1426
22
100
n.d.
n.d.
n.d.
22
200
n.d.
n.d.
n.d.
Method
Reference
HPLC-FLD
Li and Yoshizawa
(2000)
Asia
AOH
AME
China
ALT
ATX-I
TeA
EFSA Journal 2011;9(10):2407
Wheat grain
(weather
damaged)
Wheat grain
(weather
damaged)
Wheat grain
(weather
damaged)
Wheat grain
(weather
damaged)
Wheat grain
(weather
damaged)
HPLC-UV
22
22
100
2419
260
Li and Yoshizawa
(2000)
6432
93

Region/Country
Alternaria
toxin
Commodity
n>
LOQ
LOD(a)/LOQ(b)/
CC(c)/CC(d)
Mean
Minimum
Maximum
Wheat grain
(visibly affected
by Alternaria
rot)
33
10(b)
n.d.
1050
Wheat grain
10
n.d.
n.d.
n.d.
Sorghum
12
10
n.d.
n.d.
n.d.
Wheat grain
(visibly affected
by Alternaria
rot)
33
10
n.d.
46
Wheat grain
10
n.d.
n.d.
n.d.
Sorghum
Wheat grain
(visibly affected
by Alternaria
rot)
12
10
n.d.
n.d.
n.d.
33
10
n.d.
220
Wheat grain
10
n.d.
15
Sorghum
12
12
10
115
Method
Reference
HPLC-UV
Webley et al.
(1997)
Australia
AOH
Australia
AME
TeA
EFSA Journal 2011;9(10):2407
94

Region/Country
Alternaria
toxin
Commodity
n>
LOQ
LOD(a)/LOQ(b)/
CC(c)/CC(d)
Mean
Minimum
Maximum
22
18
50(b)
180
50
570
22
16
40
100
40
270
22
40
n.d.
n.d.
n.d.
22
200
n.d.
n.d.
n.d.
22
22
350
1900
500
5600
Method
Reference
HPLC-FLD
Nawaz et al. (1997)
HPLC-UV
Nawaz et al. (1997)

Nawaz et al. (1997)
Various origins
AOH
EC origin,
Argentina, India
AME
ALT
EC origin,
Argentina, India
ATX-I
TeA
Sunflower seed
meal
Sunflower seed
meal
Sunflower seed
meal
Sunflower seed
meal
Sunflower seed
meal
LOD: limit of detection; LOQ = limit of quantification; CC: decision limit; CC: detection capability; (a): LOD; (b): LOQ; (c): CC; (d): CC; (e): n of samples > LOD; AOH: alternariol;
AME: alternariol monomethyl ether; ALT: altenuene; TeA: tenuazonic acid; ATX: altertoxin; AAL-toxins: A. alternata f. sp. Lycopersici toxins; n.d.: not detected; -: not reported; LC-MS/MS:
liquid chromatographymass spectrometry; HPTLC: high performance thin layer chromatography; HPLC: high performance liquid chromatography; UV: ultraviolet detection; FLD:
fluorescence detection; DAD: diode array detection: MS: mass spectrometry; ELISA: enzyme-linked immunosorbent assay; TLC: thin layer chromatography; EC: European Community.
EFSA Journal 2011;9(10):2407
95
ABBREVIATIONS
AAL-toxins
ACR-toxin
ACTG-toxin
AESAN
AESAN_FIAB
AF-toxin
AK-toxin
ALT
AM-toxin
AME
AOH
AS-toxin
ATX
aw
BE
BfR
b.w.
CAS
CC
CC
Comprehesive Database
CONTAM Panel
CZ
DAD
Danish_Dietary_Survey
DCM
DE
Diet_National_2004
DK
DMSO
DNFCS_2003
EC
EFSA
EFSA_TEST
EI
EIA
ELISA
ER
ES
FI
FINDIET_2007
FLD
FR
GC
GPC
hCYP
HFB
HLB
HPLC
EFSA Journal 2011;9(10):2407
from Alternaria alternata f. sp. lycopersici toxins

from A. alternata rough lemon pathotype, former A. citri
from A. alternata tangerine pathotype, former A. citri
Spanish Food and Drink Industry Federation
Spanish Food and Drink Industry Federation Spanish Dietary
Survey
from A. alternata f. sp. Fragariae
from A. alternata japanese pear pathotype, former A. kikuchiana S.
Tanaka
Altenuene
From A. mali
Alternariol monomethyl ether
Alternariol
from A. alternata pathogenic to sunflower
Altertoxin
Water activity
Belgium
Bundesinstitut fr Risikobewertung/German Federal Institute for Risk
Assessment
Body weight
Chemical Abstracts Services
Decision limit
Detection capability
The EFSA Comprehensive European Food Consumption Database
EFSAs Panel on Contaminants in the Food Chain
Czech Republic
Diode array detection
Danish Dietary Survey
EFSAs Dietary and Chemical Monitoring (former DATEX) Unit
Germany
Diet_National_2004 (Belgium, Dietary survey)
Denmark
Dimethylsulphoxide
Dutch National Food Consumption Survey
European Commission
European Food Safety Authority
EFSA_TEST (Latvia, Dietary survey)
Electron impact
Enzyme immunoassay
Enzyme-linked immunosorbent assay
Estrogen receptor
Spain
Finland
FINDIET_2007 (Finland, Dietary survey)
Fluorescence detection
France
Gas chromatography
Gel permeation chromatography
Human cytochrome P450
Heptafluorobutyrates
Hydrophilic-lipophilic balance
High performance liquid chromatography
96
HU
HPTLC
hUGTs
IE
INCA2
INRAN_SCAI_2005_06
i.p.
iso-TeA
IT
i.v.
LB
LC-MS/MS
LLE
LOAEL
LOD
LOQ
LT
MS
MS/MS
MW
National_Nutrition_Survey_II
National_Repr_Surv
n.d.
NDNS
NL
n.q.
NSIFCS
P4
P450ssc
Riksmaten_1997_98
s.c.
SE
SISP04
SPE
TDP1
TeA
TEN
TLC
TK
TTC
UB
UK
UPLC
UV
EFSA Journal 2011;9(10):2407
Hungary
High performance thin layer chromatography
Human UDP-glucuronosyltransferases
Ireland
Enqute Individuelle et Nationale sur les Consommations
Alimentaires
Italian National Food Consumption Survey
Intraperitoneal
Iso-tenuazonic acid
Italy
Intravenous
Lower bound
Liquid chromatographymass spectrometry
Liquid/liquid extraction
Lowest-observed-adverse-effect-level
Limit of detection
Limit of quantification
Latvia
Mass spectrometry
Tandem mass spectrometry
Molecular weight
National_Nutrition_Survey_II (Germany, Dietary survey)
National_Repr_Surv (Hungary, Dietary survey)
Not detected
National Diet and Nutrition Survey (United Kingdom)
The Netherlands
Not quantified
North/South Ireland Food Consumption Survey
Progesterone
Cytochrome P450 side-chain cleavage enzyme
Swedish national food survey_1997_98
Subcutaneous
Sweden
SISP04 Czech dietary survey
Solid phase extraction
Tyrosyl-DNA phosphodiesterase I
Tenuazonic acid
Tentoxin
Thin-layer chromatography
Toxicokinetic
Threshold of toxicological concern
Upper bound
United Kingdom
Ultra performance liquid chromatography
Ultraviolet
97

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EFSA Journal 2011;9(10):2407

European Food Safety Authority, 2011