# Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health

Henriqueta Louro, Ariane Vettorazzi, Adela López de Cerain, Anastasia

Spyropoulou, Anita Solhaug, Anne Straumfors, Anne-Cathrin Behr, Birgit

Mertens, Bojana Žegura, Christiane Kruse Fæste, et al.

► To cite this version:

Henriqueta Louro, Ariane Vettorazzi, Adela López de Cerain, Anastasia Spyropoulou, Anita Solhaug, et al. Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health. Archives of Toxicology, 2024, 98 (2), pp.425-469. 10.1007/s00204-023-03636-8. anses-04383220

HAL Id: anses-04383220

https://anses.hal.science/anses-04383220

Submitted on 15 Jan 2024

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entific research documents, whether they are pub-
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teaching and research institutions in France or
abroad, or from public or private research centers.

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publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health

Henriqueta Louro, Ariane Vettorazzi, Adela López de Cerain, Anastasia Spyropoulou, Anita Solhaug, Anne Straumfors, Anne-Cathrin Behr, Birgit Mertens, Bojana Žegura, Christiane Kruse Fæste, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Henriqueta Louro, Ariane Vettorazzi, Adela López de Cerain, Anastasia Spyropoulou, Anita Solhaug, et al. Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health. Archives of Toxicology, 2024, 98 (2), pp.425-469. 10.1007/s00204-023-03636-8. anses-04383220

HAL Id: anses-04383220

https://anses.hal.science/anses-04383220v1

Submitted on 15 Jan 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Hazard characterization of *Alternaria* toxins to identify data gaps and improve risk assessment for human health

Henriqueta Louro¹ ID · Ariane Vettorazzi² ID · Adela López de Cerain² ID · Anastasia Spyropoulou³ ID ·

Anita Solhaug⁴ ID · Anne Straumfors⁴ ID · Anne-Cathrin Behr⁵ ID · Birgit Mertens⁶ ID · Bojana Žegura⁷ ID ·

Christiane Kruse Fæste⁴ ID · Dieynaba Ndiaye⁸ ID · Eliana Spilioti³ ID · Elisabeth Varga⁹,¹⁰ ID · Estelle Dubreil¹¹ ID ·

Eszter Borsos¹⁰ ID · Francesco Crudo¹⁰ ID · Gunnar Sundstøl Eriksen⁴ ID · Igor Snapkow¹² ID · Jérôme Henri¹¹ ID ·

Julie Sanders⁶ ID · Kyriaki Machera³ ID · Laurent Gaté⁸ ID · Ludovic Le Hegarat¹¹ ID · Matjaž Novak⁷ ID ·

Nicola M. Smith¹² ID · Solveig Krapf⁴ ID · Sonja Hager⁹ ID · Valérie Fessard¹¹ ID · Yvonne Kohl¹³ ID · Maria João Silva¹ ID ·

Hubert Dirven¹² ID · Jessica Dietrich¹⁴ ID · Doris Marko⁹ ID

Received: 30 October 2023 / Accepted: 9 November 2023

© The Author(s) 2023

## Abstract

Fungi of the genus *Alternaria* are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of *Alternaria* toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified *Alternaria* toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further *Alternaria* toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of *Alternaria* toxins. Overall, hazard characterization of *Alternaria* toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based *Alternaria* toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of *Alternaria* mycotoxins for consumers and occupationally exposed workers.

**Keywords** Mycotoxin · Exposure routes · Genotoxicity · Endocrine disruption · Immunosuppression · Biotransformation · Toxicokinetics · Tenuazonic acid · Alternariol · Altenuene · Tentoxin · Altertoxin

## Introduction

Mycotoxins are secondary metabolites produced by diverse fungi genera that contaminate food and feed worldwide. Climate conditions represent a critical factor for fungal growth and toxin expression. The anticipated climatic changes are expected to affect the geographic distribution and growth conditions of fungi, which in consequence might change the exposure pattern and increase human exposure to mycotoxins (Perrone et al. 2020).

Fungi of the genus *Alternaria* are ubiquitous plant pathogens and saprophytes that can contaminate a broad range of crops and raw materials. They produce a variety of structurally diverse secondary toxic metabolites including not only several dibenzo-α-pyrones and perylene quinones but also a spectrum of toxic metabolites with miscellaneous structures (Fig. 1). *Alternaria* mycotoxins can be found in both fresh and processed foods, including grains and grain-based products, sunflower seeds and oil, tomato and tomato products, fruits and fruit products, and fermented beverages like beer and wine (EFSA 2011). Being often heat and cold stable, and resistant to processing, boiling, fermenting, and other

<footnote>Extended author information available on the last page of the article</footnote>
# Hazard characterization of *Alternaria* toxins to identify data gaps and improve risk assessment for human health

Henriqueta Louro¹ ID · Ariane Vettorazzi² ID · Adela López de Cerain² ID · Anastasia Spyropoulou³ ID ·

Anita Solhaug⁴ ID · Anne Straumfors⁴ ID · Anne-Cathrin Behr⁵ ID · Birgit Mertens⁶ ID · Bojana Žegura⁷ ID ·

Christiane Kruse Fæste⁴ ID · Dieynaba Ndiaye⁸ ID · Eliana Spilioti³ ID · Elisabeth Varga⁹,¹⁰ ID · Estelle Dubreil¹¹ ID ·

Eszter Borsos¹⁰ ID · Francesco Crudo¹⁰ ID · Gunnar Sundstøl Eriksen⁴ ID · Igor Snapkow¹² ID · Jérôme Henri¹¹ ID ·

Julie Sanders⁶ ID · Kyriaki Machera³ ID · Laurent Gaté⁸ ID · Ludovic Le Hegarat¹¹ ID · Matjaž Novak⁷ ID ·

Nicola M. Smith¹² ID · Solveig Krapf⁴ ID · Sonja Hager⁹ ID · Valérie Fessard¹¹ ID · Yvonne Kohl¹³ ID · Maria João Silva¹ ID ·

Hubert Dirven¹² ID · Jessica Dietrich¹⁴ ID · Doris Marko⁹ ID

Received: 30 October 2023 / Accepted: 9 November 2023

© The Author(s) 2023

## Abstract

Fungi of the genus *Alternaria* are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of *Alternaria* toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified *Alternaria* toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further *Alternaria* toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of *Alternaria* toxins. Overall, hazard characterization of *Alternaria* toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based *Alternaria* toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of *Alternaria* mycotoxins for consumers and occupationally exposed workers.

**Keywords** Mycotoxin · Exposure routes · Genotoxicity · Endocrine disruption · Immunosuppression · Biotransformation · Toxicokinetics · Tenuazonic acid · Alternariol · Altenuene · Tentoxin · Altertoxin

## Introduction

Mycotoxins are secondary metabolites produced by diverse fungi genera that contaminate food and feed worldwide. Climate conditions represent a critical factor for fungal growth and toxin expression. The anticipated climatic changes are expected to affect the geographic distribution and growth conditions of fungi, which in consequence might change the exposure pattern and increase human exposure to mycotoxins (Perrone et al. 2020).

Fungi of the genus *Alternaria* are ubiquitous plant pathogens and saprophytes that can contaminate a broad range of crops and raw materials. They produce a variety of structurally diverse secondary toxic metabolites including not only several dibenzo-α-pyrones and perylene quinones but also a spectrum of toxic metabolites with miscellaneous structures (Fig. 1). *Alternaria* mycotoxins can be found in both fresh and processed foods, including grains and grain-based products, sunflower seeds and oil, tomato and tomato products, fruits and fruit products, and fermented beverages like beer and wine (EFSA 2011). Being often heat and cold stable, and resistant to processing, boiling, fermenting, and other

<footnote>Extended author information available on the last page of the article</footnote>
<figure><img src="image_2.png" /><figcaption>**Fig. 1** The structures of *Alternaria* mycotoxins: alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altenusin (ALS), altersetin (ATS), tentoxin (TEN) tenuazonic acid (TeA) and</figcaption></figure>

the perylene quinones altertoxin I, II, III (ATX-I, -II, and -III), stem-
phyltoxin III (STTX-III) and alterperylenol/alteichin (ALP)

commonly applied food processing techniques, mycotoxins
already present in the raw material tend to stay in the prod-
uct. *Alternaria* mycotoxins are also present in dust gener-
ated during occupational handling of food and feed ingredi-
ents as well as contaminated debris from raw materials and
waste for destruction. This poses an occupational hazard for
workers in agriculture, waste handling and food production
and processing through inhalation and dermal exposure to
mycotoxins (Halstensen et al. 2008; Straumfors et al. 2015;
Mayer et al. 2016; Viegas et al. 2018). Alternariol (AOH),
alternariol monomethyl ether (AME), altenuene (ALT), and
tentoxin (TEN) have been found in 76–100% of the dust
samples collected at industrial grain and animal feed mills
with mean and maximum concentrations of up to 55 and 434
µg/kg, respectively (Straumfors et al. 2015). Considering a
dust exposure of 0.03–100 mg/m³, the estimated worst-case
exposure to the detected *Alternaria* toxins would amount
to 55 ng/m³. Occurrence data for *Alternaria* mycotoxins in
food are mostly available for AOH, AME, ALT, tenuazonic
acid (TeA), and TEN, while very little information is cur-
rently available for the perylene quinones altertoxin I, II, and
III (ATX-I, -II, and -III), stemphyltoxin I and III (STTX-I
and STTX-III), and alterperylenol/alteichin (ALP). Food can
often be contaminated by multiple *Alternaria* mycotoxins,
with up to six different toxins being found in some cases,
as demonstrated exemplarily for tomato sauce, wheat flour,

and sunflower seed oil (Crudo et al. 2019; Puntscher et al. 2018). However, the occurrence data currently available are not sufficient to set maximum levels for *Alternaria* toxins in food (EFSA et al. 2016). Therefore, in 2022, the European Commission published a recommendation, in which the member states are asked to monitor the occurrence of *Alternaria* toxins in food, focusing on AOH, AME, and TeA (European Commission 2022). Processed tomato products, paprika powder, sesame seeds, sunflower seeds, sunflower oil, tree nuts, dried figs, and cereal-based foods for infants and young children are the main foodstuffs to be investigated. In addition, the recommendation includes indicative levels, but no safety levels, for AOH, AME, and TeA in the above-mentioned matrices based on current occurrence data provided by the European Food Safety Authority (EFSA). When the levels are exceeded, food manufacturers should investigate relevant input factors including *Alternaria* toxins occurrence and effects of food processing (European Commission 2022). According to the WHO, mycotoxins in indoor environments should be classified as potential health hazards, even though there is no strong evidence relating indoor mycotoxin exposure to arising diseases (WHO 2009).

In contrast to the majority of chemicals, hazard assess-
ment of natural toxins is mainly based on academic data
provided in publications. The Panel on Contaminants in the
Food Chain (CONTAM Panel) of EFSA has published two

<figure><img src="image_3.png" /></figure>
<figure><img src="image_3.png" /><figcaption>**Fig. 1** The structures of *Alternaria* mycotoxins: alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altenusin (ALS), altersetin (ATS), tentoxin (TEN) tenuazonic acid (TeA) and</figcaption></figure>

the perylene quinones altertoxin I, II, III (ATX-I, -II, and -III), stem-
phyltoxin III (STTX-III) and alterperylenol/alteichin (ALP)

commonly applied food processing techniques, mycotoxins
already present in the raw material tend to stay in the prod-
uct. *Alternaria* mycotoxins are also present in dust gener-
ated during occupational handling of food and feed ingredi-
ents as well as contaminated debris from raw materials and
waste for destruction. This poses an occupational hazard for
workers in agriculture, waste handling and food production
and processing through inhalation and dermal exposure to
mycotoxins (Halstensen et al. 2008; Straumfors et al. 2015;
Mayer et al. 2016; Viegas et al. 2018). Alternariol (AOH),
alternariol monomethyl ether (AME), altenuene (ALT), and
tentoxin (TEN) have been found in 76–100% of the dust
samples collected at industrial grain and animal feed mills
with mean and maximum concentrations of up to 55 and 434
µg/kg, respectively (Straumfors et al. 2015). Considering a
dust exposure of 0.03–100 mg/m³, the estimated worst-case
exposure to the detected *Alternaria* toxins would amount
to 55 ng/m³. Occurrence data for *Alternaria* mycotoxins in
food are mostly available for AOH, AME, ALT, tenuazonic
acid (TeA), and TEN, while very little information is cur-
rently available for the perylene quinones altertoxin I, II, and
III (ATX-I, -II, and -III), stemphyltoxin I and III (STTX-I
and STTX-III), and alterperylenol/alteichin (ALP). Food can
often be contaminated by multiple *Alternaria* mycotoxins,
with up to six different toxins being found in some cases,
as demonstrated exemplarily for tomato sauce, wheat flour,

and sunflower seed oil (Crudo et al. 2019; Puntscher et al. 2018). However, the occurrence data currently available are not sufficient to set maximum levels for *Alternaria* toxins in food (EFSA et al. 2016). Therefore, in 2022, the European Commission published a recommendation, in which the member states are asked to monitor the occurrence of *Alternaria* toxins in food, focusing on AOH, AME, and TeA (European Commission 2022). Processed tomato products, paprika powder, sesame seeds, sunflower seeds, sunflower oil, tree nuts, dried figs, and cereal-based foods for infants and young children are the main foodstuffs to be investigated. In addition, the recommendation includes indicative levels, but no safety levels, for AOH, AME, and TeA in the above-mentioned matrices based on current occurrence data provided by the European Food Safety Authority (EFSA). When the levels are exceeded, food manufacturers should investigate relevant input factors including *Alternaria* toxins occurrence and effects of food processing (European Commission 2022). According to the WHO, mycotoxins in indoor environments should be classified as potential health hazards, even though there is no strong evidence relating indoor mycotoxin exposure to arising diseases (WHO 2009).

In contrast to the majority of chemicals, hazard assess-
ment of natural toxins is mainly based on academic data
provided in publications. The Panel on Contaminants in the
Food Chain (CONTAM Panel) of EFSA has published two

<figure><img src="image_4.png" /></figure>
scientific opinions on *Alternaria* toxins. The first addressed
the risks for animal and human health related to the presence
of *Alternaria* toxins in feed and food (EFSA 2011), while the
second focused on dietary exposure assessment to *Alternaria*
toxins in the European population (EFSA et al. 2016). In
2011, the CONTAM Panel concluded that the toxicologi-
cal data were not sufficient to establish risk-based guidance
values. Instead, TTC values (threshold of toxicological con-
cern) were assigned to, respectively, AOH, AME, TeA and
TEN, as described in the "Scientific Opinion on Exploring
options for providing advice about possible human health
risks based on the concept of Threshold of Toxicological
Concern (TTC)" (EFSA 2011; EFSA Scientific Committee
2012). The concept is used in cases, where only scarce toxi-
cological data are available, but nonetheless a risk evaluation
of chronic exposure is required. It allows for an assessment
of the chronic exposure of substances with known structure
but with incomplete hazard data. TTC values are not risk-
based guidance values, but threshold values for exposure.
If the exposure in humans is consistently higher than the
TTC, the need for additional toxicity data is immediate as a
prerequisite for a comprehensive risk assessment. At expo-
sure below the TTC, a negligible risk to human health is
assumed (EFSA Scientific Committee 2012; EFSA Scien-
tific Committee et al. 2019). For potentially mutagenic and/
or carcinogenic substances—such as AOH and AME—the
TTC for chronic dietary exposure has been set to 2.5 ng/
kg b.w. per day. For TeA and TEN, there is no evidence
of genotoxic potential so far. Therefore, the assigned TTC
has been set to 1.5 µg/kg b.w. per day. No TTC value was
assigned to ALT and the other *Alternaria* toxins because
the data were insufficient for the application of the TTC
concept by EFSA's CONTAM Panel. Comparison to the esti-
mated mean dietary intakes in European adults indicated
that the TTC are frequently exceeded, at least for AOH and
AME. Consideration of 95th-percentile exposure indicates
that there is a notable risk to human health from these two
*Alternaria* toxins in food in Europe. It is worth mentioning
that the presence of *Alternaria* mycotoxins in food might
be underestimated in current estimates, as some mycotox-
ins could be lost during filtration in the sample preparation
process for mycotoxin quantification, as previously reported
(Aichinger et al. 2020a). The occupational exposure through
skin and inhalation may add considerably to the dietary
exposure for workers employed in the food production indus-
try. This underlines that further research in terms of hazard
identification and characterization is required for both toxins
(EFSA 2011; EFSA et al. 2016). Besides this, major data
gaps regarding the toxicity of *Alternaria* toxins were also
identified in a report by the Norwegian Scientific Committee
for Food and the Environment (VKM et al. 2019). Moreo-
ver, an increasing number of studies show co-occurrence
of *Alternaria* toxins and other xenobiotics in food (Crudo

et al. 2019). For this reason, not only the consideration of
the individual components but also mixtures are required for
hazard characterization.

The aim of this review paper is to summarize the state
of research regarding the hazard identification and hazard
characterization of *Alternaria* toxins to identify data gaps
to improve risk assessment for human health by additional
testing. Endpoints such as cytotoxicity, genotoxicity, immu-
notoxicity, endocrine effects and toxicokinetics, both in vitro
and in vivo, are considered to provide an overview of exist-
ing toxicological data under the scope of the European Part-
nership for the Assessment of Risks from Chemicals (PARC,
https://www.eu-parc.eu), identifying further data needs.

Cytotoxicity

Numerous studies have investigated the cytotoxicity of *Alternaria* toxins. Data on cytotoxicity are available for ALP, ALS, ALT, AME, AOH, ATX-II, STTX-II, TeA, TEN as well as different mixtures on a broad panel of cell types, over a treatment duration up to 72 h (Table 1). Since the epithelium of the gastrointestinal tract (GIT) is a crucial barrier against foodborne xenobiotics and the main site of nutrient absorption, GIT cell models are essential in vitro systems to investigate *Alternaria* toxin uptake and toxicity. In addition to the GIT, other biological barriers, such as skin and lung, but also a variety of other organs were investigated for *Alternaria* toxicity. The toxins have been tested in human epithelial cell lines: four from intestine (Caco-2, HT29, HCEC-1CT, HCT116) and three from lung (A549, BEAS-2B, NCIH460), as well in Chinese hamster lung fibroblast cells (V79). Moreover, cell lines from liver, skin, kidney, ovary, prostate, mammary, brain, cervix, uterus, esophagus, and adrenal gland as well as two types of monocytes have been used.

ªLow concentration chosen intentionally because it was equal to 5 exceeding the recommended daily dose under conditions of 100% bioavailability (Tran et al. 2020).

Individual toxins

AOH is the most studied *Alternaria* toxin, showing cyto-
toxic effects in almost all studies performed. The most
used intestinal cell lines in *Alternaria* in vitro toxicity
studies are Caco-2 (tight junction-forming human colon
cancer cell line, when differentiated) and HT29 (epithelial
human colon cancer cell line, depending on the respec-
tive strain). Exposure of non-differentiated Caco-2 cells
to 3.125–100 µM AOH for 24 h resulted in significant
cytotoxicity at 25 µM and 50 µM, but it was not possible to
determine IC₅₀ (Vila-Donat et al. 2015). Cytotoxic effects
were detected neither in IL-1β-stimulated differentiated
scientific opinions on *Alternaria* toxins. The first addressed
the risks for animal and human health related to the presence
of *Alternaria* toxins in feed and food (EFSA 2011), while the
second focused on dietary exposure assessment to *Alternaria*
toxins in the European population (EFSA et al. 2016). In
2011, the CONTAM Panel concluded that the toxicologi-
cal data were not sufficient to establish risk-based guidance
values. Instead, TTC values (threshold of toxicological con-
cern) were assigned to, respectively, AOH, AME, TeA and
TEN, as described in the "Scientific Opinion on Exploring
options for providing advice about possible human health
risks based on the concept of Threshold of Toxicological
Concern (TTC)" (EFSA 2011; EFSA Scientific Committee
2012). The concept is used in cases, where only scarce toxi-
cological data are available, but nonetheless a risk evaluation
of chronic exposure is required. It allows for an assessment
of the chronic exposure of substances with known structure
but with incomplete hazard data. TTC values are not risk-
based guidance values, but threshold values for exposure.
If the exposure in humans is consistently higher than the
TTC, the need for additional toxicity data is immediate as a
prerequisite for a comprehensive risk assessment. At expo-
sure below the TTC, a negligible risk to human health is
assumed (EFSA Scientific Committee 2012; EFSA Scien-
tific Committee et al. 2019). For potentially mutagenic and/
or carcinogenic substances—such as AOH and AME—the
TTC for chronic dietary exposure has been set to 2.5 ng/
kg b.w. per day. For TeA and TEN, there is no evidence
of genotoxic potential so far. Therefore, the assigned TTC
has been set to 1.5 µg/kg b.w. per day. No TTC value was
assigned to ALT and the other *Alternaria* toxins because
the data were insufficient for the application of the TTC
concept by EFSA's CONTAM Panel. Comparison to the esti-
mated mean dietary intakes in European adults indicated
that the TTC are frequently exceeded, at least for AOH and
AME. Consideration of 95th-percentile exposure indicates
that there is a notable risk to human health from these two
*Alternaria* toxins in food in Europe. It is worth mentioning
that the presence of *Alternaria* mycotoxins in food might
be underestimated in current estimates, as some mycotox-
ins could be lost during filtration in the sample preparation
process for mycotoxin quantification, as previously reported
(Aichinger et al. 2020a). The occupational exposure through
skin and inhalation may add considerably to the dietary
exposure for workers employed in the food production indus-
try. This underlines that further research in terms of hazard
identification and characterization is required for both toxins
(EFSA 2011; EFSA et al. 2016). Besides this, major data
gaps regarding the toxicity of *Alternaria* toxins were also
identified in a report by the Norwegian Scientific Committee
for Food and the Environment (VKM et al. 2019). Moreo-
ver, an increasing number of studies show co-occurrence
of *Alternaria* toxins and other xenobiotics in food (Crudo

et al. 2019). For this reason, not only the consideration of
the individual components but also mixtures are required for
hazard characterization.

The aim of this review paper is to summarize the state
of research regarding the hazard identification and hazard
characterization of *Alternaria* toxins to identify data gaps
to improve risk assessment for human health by additional
testing. Endpoints such as cytotoxicity, genotoxicity, immu-
notoxicity, endocrine effects and toxicokinetics, both in vitro
and in vivo, are considered to provide an overview of exist-
ing toxicological data under the scope of the European Part-
nership for the Assessment of Risks from Chemicals (PARC,
https://www.eu-parc.eu), identifying further data needs.

Cytotoxicity

Numerous studies have investigated the cytotoxicity of *Alternaria* toxins. Data on cytotoxicity are available for ALP, ALS, ALT, AME, AOH, ATX-II, STTX-II, TeA, TEN as well as different mixtures on a broad panel of cell types, over a treatment duration up to 72 h (Table 1). Since the epithelium of the gastrointestinal tract (GIT) is a crucial barrier against foodborne xenobiotics and the main site of nutrient absorption, GIT cell models are essential in vitro systems to investigate *Alternaria* toxin uptake and toxicity. In addition to the GIT, other biological barriers, such as skin and lung, but also a variety of other organs were investigated for *Alternaria* toxicity. The toxins have been tested in human epithelial cell lines: four from intestine (Caco-2, HT29, HCEC-1CT, HCT116) and three from lung (A549, BEAS-2B, NCIH460), as well in Chinese hamster lung fibroblast cells (V79). Moreover, cell lines from liver, skin, kidney, ovary, prostate, mammary, brain, cervix, uterus, esophagus, and adrenal gland as well as two types of monocytes have been used.

ªLow concentration chosen intentionally because it was equal to 5 exceeding the recommended daily dose under conditions of 100% bioavailability (Tran et al. 2020).

Individual toxins

AOH is the most studied *Alternaria* toxin, showing cyto-
toxic effects in almost all studies performed. The most
used intestinal cell lines in *Alternaria* in vitro toxicity
studies are Caco-2 (tight junction-forming human colon
cancer cell line, when differentiated) and HT29 (epithelial
human colon cancer cell line, depending on the respec-
tive strain). Exposure of non-differentiated Caco-2 cells
to 3.125–100 µM AOH for 24 h resulted in significant
cytotoxicity at 25 µM and 50 µM, but it was not possible to
determine IC₅₀ (Vila-Donat et al. 2015). Cytotoxic effects
were detected neither in IL-1β-stimulated differentiated
<table><caption>**Table 1** Summary of reported cytotoxicity studies on Alternaria toxins</caption><thead><tr><th>sToxin</th><th>Cell type</th><th>Cell model</th><th>Assay</th><th>Dose (µM)</th><th>Exposure time (h)</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>AOH</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2, differentiated</td><td>AB</td><td>0.02, 0.2, 2, 20, 40</td><td>5, 20</td><td>No effect</td><td>Schmutz et al. (2019)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>MTT</td><td>3.125, 6.25, 12.5, 25, 50, 100</td><td>24</td><td>↓ proliferation, dose dependent</td><td>Vila-Donat et al. (2015)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>PC</td><td>3.125, 6.25, 12.5, 25, 50, 10</td><td>24</td><td>↓ protein content ≥ 25 µM, no IC<sub>50</sub></td><td>Vila-Donat et al. (2015)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>MTT</td><td>1.85, 3.1, 7.5, 15, 30, 60, 90</td><td>24, 48, 72</td><td>↓ viability ≥ 60 µM, no IC<sub>50</sub><br>Combinatory effects with enniatins</td><td>Fernández-Blanco et al. (2016b)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>MTT</td><td>12.5, 25, 37.5, 50, 75, 100</td><td>24, 48, 72</td><td>↓ proliferation, dose dependent</td><td>Chiesi et al. (2015)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>MTT</td><td>3.125–100</td><td>24, 48</td><td>↓ viability (48 h), no IC<sub>50</sub></td><td>Fernández-Blanco et al. (2016a)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> up to 50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>FC</td><td>0.4, 1, 1.9, 3.9, 19.4, 38.7, 77.5, 232.4, 464.7</td><td>48</td><td>EC<sub>50</sub> 72.5 µM</td><td>den Hollander et al. (2022)</td></tr><tr><td>AOH</td><td>Colon</td><td>HT29</td><td>WST-1</td><td>5, 7.5, 10, 25, 50, 75, 100, 200</td><td>24</td><td>No EC<sub>50</sub>, combinatory effect with ATX-II</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>AOH</td><td>Colon</td><td>HT29</td><td>SRB</td><td>0.01, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 25, 50</td><td>24</td><td>Dose dependent ↓ > 5 µM</td><td>Tiessen et al. (2013a)</td></tr><tr><td>AOH</td><td>Colon</td><td>HT29</td><td>WST-1</td><td>1, 10, 25, 50, 100</td><td>24</td><td>Effect > 25 µM</td><td>Aichinger et al. (2017)</td></tr><tr><td>AOH</td><td>Colon</td><td>HT29</td><td>SRB</td><td>1, 10, 25, 50, 100</td><td>24</td><td>Effect > 50 µM</td><td>Aichinger et al. (2017)</td></tr><tr><td>AOH</td><td>Colon</td><td>HT29</td><td>LDH</td><td>0–50</td><td>1</td><td>No effect</td><td>Fehr et al. (2009)</td></tr><tr><td>AOH</td><td>Colon</td><td>HCT116</td><td>FDA</td><td>10, 25, 50, 100, 200</td><td>24</td><td>IC<sub>50</sub> 65 µM</td><td>Bensassi et al. (2012)</td></tr><tr><td>AOH</td><td>Colon</td><td>HCEC-1CT</td><td>WST-1</td><td>0.1, 0.5, 1, 2.5, 5, 7.5, 10, 25, 50, 75, 100, 200</td><td>24</td><td>EC<sub>50</sub> 100.9 µM, combinatory effect with ATX-II</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>AOH</td><td>Lung</td><td>A549</td><td>AB</td><td>no info</td><td>72</td><td>EC<sub>50</sub> 2.69 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>AOH</td><td>Lung</td><td>BEAS-2B</td><td>MTT</td><td>1, 5, 10, 15, 20, 25, 30, 50, 100</td><td>24</td><td>↓ cell proliferation, dose dependent > 1 µM</td><td>Grover and Lawrence (2017)</td></tr><tr><td>AOH</td><td>Lung</td><td>V79</td><td>CC</td><td>5, 10, 15, 20</td><td>24</td><td>Accumulation in G2/M phase > 10 µM;</td><td>Fleck et al. (2012)</td></tr><tr><td>AOH</td><td>Lung</td><td>V79</td><td>FC</td><td>10, 20, 30</td><td>24</td><td>↑ cells in G2/M phase dose dependent</td><td>Brugger et al. (2006)</td></tr><tr><td>AOH</td><td>Lung</td><td>V79</td><td>FC</td><td>5, 10, 25, 50</td><td>6</td><td>Dose-dependent effect ↑ cell number in S and G2/M phase</td><td>Lehmann et al. (2006)</td></tr></tbody></table>
<table><caption>**Table 1** Summary of reported cytotoxicity studies on Alternaria toxins</caption><thead><tr><th>sToxin</th><th>Cell type</th><th>Cell model</th><th>Assay</th><th>Dose (µM)</th><th>Exposure time (h)</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>AOH</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2, differentiated</td><td>AB</td><td>0.02, 0.2, 2, 20, 40</td><td>5, 20</td><td>No effect</td><td>Schmutz et al. (2019)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>MTT</td><td>3.125, 6.25, 12.5, 25, 50, 100</td><td>24</td><td>↓ proliferation, dose dependent</td><td>Vila-Donat et al. (2015)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>PC</td><td>3.125, 6.25, 12.5, 25, 50, 10</td><td>24</td><td>↓ protein content ≥ 25 µM, no IC<sub>50</sub></td><td>Vila-Donat et al. (2015)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>MTT</td><td>1.85, 3.1, 7.5, 15, 30, 60, 90</td><td>24, 48, 72</td><td>↓ viability ≥ 60 µM, no IC<sub>50</sub><br>Combinatory effects with enniatins</td><td>Fernández-Blanco et al. (2016b)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>MTT</td><td>12.5, 25, 37.5, 50, 75, 100</td><td>24, 48, 72</td><td>↓ proliferation, dose dependent</td><td>Chiesi et al. (2015)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>MTT</td><td>3.125–100</td><td>24, 48</td><td>↓ viability (48 h), no IC<sub>50</sub></td><td>Fernández-Blanco et al. (2016a)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> up to 50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>FC</td><td>0.4, 1, 1.9, 3.9, 19.4, 38.7, 77.5, 232.4, 464.7</td><td>48</td><td>EC<sub>50</sub> 72.5 µM</td><td>den Hollander et al. (2022)</td></tr><tr><td>AOH</td><td>Colon</td><td>HT29</td><td>WST-1</td><td>5, 7.5, 10, 25, 50, 75, 100, 200</td><td>24</td><td>No EC<sub>50</sub>, combinatory effect with ATX-II</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>AOH</td><td>Colon</td><td>HT29</td><td>SRB</td><td>0.01, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 25, 50</td><td>24</td><td>Dose dependent ↓ > 5 µM</td><td>Tiessen et al. (2013a)</td></tr><tr><td>AOH</td><td>Colon</td><td>HT29</td><td>WST-1</td><td>1, 10, 25, 50, 100</td><td>24</td><td>Effect > 25 µM</td><td>Aichinger et al. (2017)</td></tr><tr><td>AOH</td><td>Colon</td><td>HT29</td><td>SRB</td><td>1, 10, 25, 50, 100</td><td>24</td><td>Effect > 50 µM</td><td>Aichinger et al. (2017)</td></tr><tr><td>AOH</td><td>Colon</td><td>HT29</td><td>LDH</td><td>0–50</td><td>1</td><td>No effect</td><td>Fehr et al. (2009)</td></tr><tr><td>AOH</td><td>Colon</td><td>HCT116</td><td>FDA</td><td>10, 25, 50, 100, 200</td><td>24</td><td>IC<sub>50</sub> 65 µM</td><td>Bensassi et al. (2012)</td></tr><tr><td>AOH</td><td>Colon</td><td>HCEC-1CT</td><td>WST-1</td><td>0.1, 0.5, 1, 2.5, 5, 7.5, 10, 25, 50, 75, 100, 200</td><td>24</td><td>EC<sub>50</sub> 100.9 µM, combinatory effect with ATX-II</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>AOH</td><td>Lung</td><td>A549</td><td>AB</td><td>no info</td><td>72</td><td>EC<sub>50</sub> 2.69 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>AOH</td><td>Lung</td><td>BEAS-2B</td><td>MTT</td><td>1, 5, 10, 15, 20, 25, 30, 50, 100</td><td>24</td><td>↓ cell proliferation, dose dependent > 1 µM</td><td>Grover and Lawrence (2017)</td></tr><tr><td>AOH</td><td>Lung</td><td>V79</td><td>CC</td><td>5, 10, 15, 20</td><td>24</td><td>Accumulation in G2/M phase > 10 µM;</td><td>Fleck et al. (2012)</td></tr><tr><td>AOH</td><td>Lung</td><td>V79</td><td>FC</td><td>10, 20, 30</td><td>24</td><td>↑ cells in G2/M phase dose dependent</td><td>Brugger et al. (2006)</td></tr><tr><td>AOH</td><td>Lung</td><td>V79</td><td>FC</td><td>5, 10, 25, 50</td><td>6</td><td>Dose-dependent effect ↑ cell number in S and G2/M phase</td><td>Lehmann et al. (2006)</td></tr></tbody></table>

<figure><img src="image_5.png" /></figure>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>sToxin</th><th>Cell type</th><th>Cell model</th><th>Assay</th><th>Dose (µM)</th><th>Exposure time (h)</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>FC</td><td>0.4, 1, 1.9, 3.9, 19.4, 38.7, 77.5, 232.4, 464.7</td><td>24</td><td>EC<sub>50</sub> 45.23 µM</td><td>den Hollander et al. (2022)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>Significant ↓ at 100 µM</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> up to 50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>AB</td><td>48.4, 96.8, 193.6, 387.3</td><td>no info</td><td>EC<sub>50</sub> 108.4 µM)</td><td>Mahmoud et al. (2022)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>3.2, 6.4, 12.8, 24</td><td>24, 48, 72</td><td>↓ cell viability > 12.8 µM combinatory effect with deoxynivalenol</td><td>Juan-García et al. (2016)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>WST-1</td><td>5, 7.5, 10, 25, 50, 75, 100, 200</td><td>24</td><td>EC<sub>50</sub> 51.4 µM, combinatory effect with ATX-II</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepaRG</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>significant ↓ at 100 µM</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>AOH</td><td>Skin</td><td>A431</td><td>LDH</td><td>0–50</td><td>1</td><td>No effect (data not shown)</td><td>Fehr et al. (2009)</td></tr><tr><td>AOH</td><td>Kidney</td><td>HEK 293 T</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> values up to 50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AOH</td><td>Mammary</td><td>184A1</td><td>AB</td><td>0.001, 0.01, 0.1, 1, 10, 30, 50, 70, 100</td><td>24, 48</td><td>Effect > 1 µM</td><td>Kowalska et al. (2021a)</td></tr><tr><td>AOH</td><td>Mammary</td><td>RGA</td><td>MTT</td><td>0.0004, 0.0039, 0.0387, 0.387, 3.87, 38.7</td><td>48</td><td>↓ viability > 19.4 µM in TARM-Luc; no effect in MMV-Luc cells</td><td>Frizzell et al. (2013)</td></tr><tr><td>AOH</td><td>Cervix</td><td>HeLa</td><td>ATP</td><td>0–250</td><td>24</td><td>IC<sub>50</sub> 33.6 µM, synergistic effect with zearalenone</td><td>Balázs et al. (2021)</td></tr><tr><td>AOH</td><td>Esophagus</td><td>KYSE510</td><td>WST-1</td><td>0.1, 1, 5, 10, 25, 50</td><td>24</td><td>57% cell viability ↓ viability at 25 and 50 µM</td><td>Tiessen et al. (2017)</td></tr><tr><td>AOH</td><td>Uterus</td><td>Ishikawa</td><td>NR</td><td>0.1, 0.5, 1, 5, 10</td><td>48</td><td>effect ≥ 5 µM</td><td>Aichinger et al. (2020b)</td></tr><tr><td>AOH</td><td>Uterus</td><td>Ishikawa</td><td>FC</td><td>0.5, 1, 2.5, 5, 10</td><td>48, 72</td><td>EC<sub>50</sub> 10 µM dose-dependent effect</td><td>Lehmann et al. (2006)</td></tr><tr><td>AOH</td><td>Ovary</td><td>PGC</td><td>MTT</td><td>0.8, 1.6, 3.2, 6.4, 12.8</td><td>24</td><td>Dose-dependent effect</td><td>Tiemann et al. (2009)</td></tr><tr><td>AOH</td><td>Ovary</td><td>CHO-K1</td><td>ATP, MTT</td><td>0–250</td><td>24</td><td>Individual and combined with genistein effects</td><td>Balázs et al. (2021)</td></tr><tr><td>AOH</td><td>Prostate</td><td>PC3</td><td>AB</td><td>No info</td><td>72</td><td>EC<sub>50</sub> 0.64 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>AOH</td><td>Prostate</td><td>PNT1A</td><td>AB</td><td>0.001, 0.01, 0.1, 1, 10, 30, 50, 70, 100</td><td>24, 48</td><td>↓ cell viability > 10 µM</td><td>Kowalska et al. (2021b)</td></tr><tr><td>AOH</td><td>Adrenal gland</td><td>H295R</td><td>AB</td><td>0.0004, 0.0039, 0.0387, 0.387, 3.87</td><td>48</td><td>No effects</td><td>Frizzell et al. (2013)</td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>sToxin</th><th>Cell type</th><th>Cell model</th><th>Assay</th><th>Dose (µM)</th><th>Exposure time (h)</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>FC</td><td>0.4, 1, 1.9, 3.9, 19.4, 38.7, 77.5, 232.4, 464.7</td><td>24</td><td>EC<sub>50</sub> 45.23 µM</td><td>den Hollander et al. (2022)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>Significant ↓ at 100 µM</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> up to 50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>AB</td><td>48.4, 96.8, 193.6, 387.3</td><td>no info</td><td>EC<sub>50</sub> 108.4 µM)</td><td>Mahmoud et al. (2022)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>3.2, 6.4, 12.8, 24</td><td>24, 48, 72</td><td>↓ cell viability > 12.8 µM combinatory effect with deoxynivalenol</td><td>Juan-García et al. (2016)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepG2</td><td>WST-1</td><td>5, 7.5, 10, 25, 50, 75, 100, 200</td><td>24</td><td>EC<sub>50</sub> 51.4 µM, combinatory effect with ATX-II</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>AOH</td><td>Liver</td><td>HepaRG</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>significant ↓ at 100 µM</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>AOH</td><td>Skin</td><td>A431</td><td>LDH</td><td>0–50</td><td>1</td><td>No effect (data not shown)</td><td>Fehr et al. (2009)</td></tr><tr><td>AOH</td><td>Kidney</td><td>HEK 293 T</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> values up to 50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AOH</td><td>Mammary</td><td>184A1</td><td>AB</td><td>0.001, 0.01, 0.1, 1, 10, 30, 50, 70, 100</td><td>24, 48</td><td>Effect > 1 µM</td><td>Kowalska et al. (2021a)</td></tr><tr><td>AOH</td><td>Mammary</td><td>RGA</td><td>MTT</td><td>0.0004, 0.0039, 0.0387, 0.387, 3.87, 38.7</td><td>48</td><td>↓ viability > 19.4 µM in TARM-Luc; no effect in MMV-Luc cells</td><td>Frizzell et al. (2013)</td></tr><tr><td>AOH</td><td>Cervix</td><td>HeLa</td><td>ATP</td><td>0–250</td><td>24</td><td>IC<sub>50</sub> 33.6 µM, synergistic effect with zearalenone</td><td>Balázs et al. (2021)</td></tr><tr><td>AOH</td><td>Esophagus</td><td>KYSE510</td><td>WST-1</td><td>0.1, 1, 5, 10, 25, 50</td><td>24</td><td>57% cell viability ↓ viability at 25 and 50 µM</td><td>Tiessen et al. (2017)</td></tr><tr><td>AOH</td><td>Uterus</td><td>Ishikawa</td><td>NR</td><td>0.1, 0.5, 1, 5, 10</td><td>48</td><td>effect ≥ 5 µM</td><td>Aichinger et al. (2020b)</td></tr><tr><td>AOH</td><td>Uterus</td><td>Ishikawa</td><td>FC</td><td>0.5, 1, 2.5, 5, 10</td><td>48, 72</td><td>EC<sub>50</sub> 10 µM dose-dependent effect</td><td>Lehmann et al. (2006)</td></tr><tr><td>AOH</td><td>Ovary</td><td>PGC</td><td>MTT</td><td>0.8, 1.6, 3.2, 6.4, 12.8</td><td>24</td><td>Dose-dependent effect</td><td>Tiemann et al. (2009)</td></tr><tr><td>AOH</td><td>Ovary</td><td>CHO-K1</td><td>ATP, MTT</td><td>0–250</td><td>24</td><td>Individual and combined with genistein effects</td><td>Balázs et al. (2021)</td></tr><tr><td>AOH</td><td>Prostate</td><td>PC3</td><td>AB</td><td>No info</td><td>72</td><td>EC<sub>50</sub> 0.64 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>AOH</td><td>Prostate</td><td>PNT1A</td><td>AB</td><td>0.001, 0.01, 0.1, 1, 10, 30, 50, 70, 100</td><td>24, 48</td><td>↓ cell viability > 10 µM</td><td>Kowalska et al. (2021b)</td></tr><tr><td>AOH</td><td>Adrenal gland</td><td>H295R</td><td>AB</td><td>0.0004, 0.0039, 0.0387, 0.387, 3.87</td><td>48</td><td>No effects</td><td>Frizzell et al. (2013)</td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>sToxin</th><th>Cell type</th><th>Cell model</th><th>Assay</th><th>Dose (µM)</th><th>Exposure time (h)</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>AOH</td><td>Macrophage</td><td>RAW264.7</td><td>AB</td><td>1–100</td><td>24, 48</td><td>IC<sub>50</sub> 49.65 µM</td><td>Solhaug et al. (2012)</td></tr><tr><td>AOH</td><td>Macrophage</td><td>RAW264.7</td><td>NR</td><td>1–100</td><td>24, 48</td><td>IC<sub>50</sub> 78.01 µM</td><td>Solhaug et al. (2012)</td></tr><tr><td>AOH</td><td>Macrophage</td><td>RAW264.7</td><td>FC</td><td>15, 30, 60</td><td>6, 24, 48</td><td>↑ necrosis > 60 µM<br>↑ apoptosis > 30 µM</td><td>Solhaug et al. (2012)</td></tr><tr><td>AOH</td><td>Monocyte</td><td>THP1</td><td>AB</td><td>10–20, 30, 60</td><td>24</td><td>Dose-dependent effect</td><td>Solhaug et al. (2016a, b)</td></tr><tr><td>AOH</td><td>Monocyte</td><td>THP1</td><td>A/N</td><td>10–20, 30, 60</td><td>24</td><td>Minor effect</td><td>Solhaug et al. (2016a, b)</td></tr><tr><td>AOH</td><td>Macrophage</td><td>THP1-Lucia™<br>NF-κBs</td><td>AB</td><td>0.02, 0.2, 2, 20</td><td>5, 20</td><td>LPS-stimulated: ↓<br>effect at 20 µM<br>Non-stimulated: no effect</td><td>Kollarova et al. (2018)</td></tr><tr><td>Alp</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ALP</td><td>Colon</td><td>HCT116</td><td>SRB</td><td>0.625, 1.25, 2.5,<br>5, 10, 20</td><td>no info</td><td>IC<sub>50</sub> 2.4 µM</td><td>Zhao et al. (2019)</td></tr><tr><td>ALP</td><td>Lung</td><td>A549</td><td>SRB</td><td>0.625, 1.25, 2.5,<br>5, 10, 20</td><td>no info</td><td>IC<sub>50</sub> 2.6 µM</td><td>Zhao et al. (2019)</td></tr><tr><td>ALP</td><td>Lung</td><td>NCIH460</td><td>SRB</td><td>No info</td><td>no info</td><td>IC<sub>50</sub> 5.47 µM</td><td>Wang et al. (2017)</td></tr><tr><td>ALP</td><td>Liver</td><td>HepG2</td><td>SRB</td><td>No info</td><td>no info</td><td>IC<sub>50</sub> 5.3 µM</td><td>Wang et al. (2017)</td></tr><tr><td>ALP</td><td>Mammary</td><td>MCF7</td><td>SRB</td><td>No info</td><td>no info</td><td>IC<sub>50</sub> 3.73 µM</td><td>Wang et al. (2017)</td></tr><tr><td>ALP</td><td>Cervix</td><td>HeLa</td><td>MTT</td><td>0.625, 1.25, 2.5,<br>5, 10, 20</td><td>no info</td><td>IC<sub>50</sub> 3.1 µM</td><td>Zhao et al. (2019)</td></tr><tr><td>ALP</td><td>Brain</td><td>SF-268</td><td>SRB</td><td>No info</td><td>no info</td><td>IC<sub>50</sub> 6.57 µM</td><td>Wang et al. (2017)</td></tr><tr><td>ALS</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ALS</td><td>Colon</td><td>HCT116</td><td>SRB</td><td>No info</td><td>72</td><td>IC<sub>50</sub> 28.9 µM</td><td>Xiao et al. (2014)</td></tr><tr><td>ALS</td><td>Skin</td><td>HaCat</td><td>MTT</td><td>5, 10, 20, 40</td><td>24</td><td>effect > 40 µM</td><td>Dong et al. (2021)</td></tr><tr><td>ALS</td><td>Brain</td><td>BV2</td><td>MTT</td><td>0–100</td><td>24</td><td>no effect</td><td>Kumar et al. (2019)</td></tr><tr><td>ALT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ALT</td><td>Colon</td><td>HCT116</td><td>SRB</td><td>No info</td><td>72</td><td>IC<sub>50</sub> 3.13 µM</td><td>Xiao et al. (2014)</td></tr><tr><td>ALT</td><td>Skin</td><td>HaCat</td><td>MTT</td><td>10, 20, 40, 80</td><td>24</td><td>No effect</td><td>Dong et al. (2021)</td></tr><tr><td>AME</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>AME</td><td>Colon</td><td>Caco-2 undif-<br>ferentiated</td><td>MTT</td><td>3.125–100</td><td>24, 48</td><td>↓ viability (48 h),<br>no IC<sub>50</sub></td><td>Fernández-Blanco<br>et al. (2016a)</td></tr><tr><td>AME</td><td>Colon</td><td>Caco-2 undif-<br>ferentiated</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> up to<br>50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AME</td><td>Colon</td><td>Caco-2 undif-<br>ferentiated</td><td>FC</td><td>0.4, 0.9, 1.8,<br>3.7, 18.4,<br>36.7, 73.5,<br>220.4, 440.8</td><td>48</td><td>EC<sub>50</sub> 56.5 µM)</td><td>den Hollander<br>et al. (2022)</td></tr><tr><td>AME</td><td>Colon</td><td>HT29</td><td>LDH</td><td>0–50</td><td>1</td><td>No effect (data not<br>shown)</td><td>Fehr et al. (2009)</td></tr><tr><td>AME</td><td>Colon</td><td>HCT116</td><td>FDA</td><td>10, 25, 50,<br>100, 200</td><td>24</td><td>IC<sub>50</sub> 120 µM</td><td>Bensassi et al. (2011)</td></tr><tr><td>AME</td><td>Lung</td><td>V79</td><td>CC</td><td>10, 20, 30, 40</td><td>24</td><td>Accumulation in G2/M<br>phase > 10 µM</td><td>Fleck et al. (2012)</td></tr><tr><td>AME</td><td>Liver</td><td>HepG2</td><td>FC</td><td>0.4, 0.9, 1.8,<br>3.7, 18.4,<br>36.7, 73.5,<br>220.4, 440.8</td><td>24</td><td>EC<sub>50</sub> 18.6 µM</td><td>den Hollander<br>et al. (2022)</td></tr></tbody></table>

<figure><img src="image_4.png" /></figure>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>sToxin</th><th>Cell type</th><th>Cell model</th><th>Assay</th><th>Dose (µM)</th><th>Exposure time (h)</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>AOH</td><td>Macrophage</td><td>RAW264.7</td><td>AB</td><td>1–100</td><td>24, 48</td><td>IC<sub>50</sub> 49.65 µM</td><td>Solhaug et al. (2012)</td></tr><tr><td>AOH</td><td>Macrophage</td><td>RAW264.7</td><td>NR</td><td>1–100</td><td>24, 48</td><td>IC<sub>50</sub> 78.01 µM</td><td>Solhaug et al. (2012)</td></tr><tr><td>AOH</td><td>Macrophage</td><td>RAW264.7</td><td>FC</td><td>15, 30, 60</td><td>6, 24, 48</td><td>↑ necrosis > 60 µM<br>↑ apoptosis > 30 µM</td><td>Solhaug et al. (2012)</td></tr><tr><td>AOH</td><td>Monocyte</td><td>THP1</td><td>AB</td><td>10–20, 30, 60</td><td>24</td><td>Dose-dependent effect</td><td>Solhaug et al. (2016a, b)</td></tr><tr><td>AOH</td><td>Monocyte</td><td>THP1</td><td>A/N</td><td>10–20, 30, 60</td><td>24</td><td>Minor effect</td><td>Solhaug et al. (2016a, b)</td></tr><tr><td>AOH</td><td>Macrophage</td><td>THP1-Lucia™<br>NF-κBs</td><td>AB</td><td>0.02, 0.2, 2, 20</td><td>5, 20</td><td>LPS-stimulated: ↓<br>effect at 20 µM<br>Non-stimulated: no effect</td><td>Kollarova et al. (2018)</td></tr><tr><td>Alp</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ALP</td><td>Colon</td><td>HCT116</td><td>SRB</td><td>0.625, 1.25, 2.5,<br>5, 10, 20</td><td>no info</td><td>IC<sub>50</sub> 2.4 µM</td><td>Zhao et al. (2019)</td></tr><tr><td>ALP</td><td>Lung</td><td>A549</td><td>SRB</td><td>0.625, 1.25, 2.5,<br>5, 10, 20</td><td>no info</td><td>IC<sub>50</sub> 2.6 µM</td><td>Zhao et al. (2019)</td></tr><tr><td>ALP</td><td>Lung</td><td>NCIH460</td><td>SRB</td><td>No info</td><td>no info</td><td>IC<sub>50</sub> 5.47 µM</td><td>Wang et al. (2017)</td></tr><tr><td>ALP</td><td>Liver</td><td>HepG2</td><td>SRB</td><td>No info</td><td>no info</td><td>IC<sub>50</sub> 5.3 µM</td><td>Wang et al. (2017)</td></tr><tr><td>ALP</td><td>Mammary</td><td>MCF7</td><td>SRB</td><td>No info</td><td>no info</td><td>IC<sub>50</sub> 3.73 µM</td><td>Wang et al. (2017)</td></tr><tr><td>ALP</td><td>Cervix</td><td>HeLa</td><td>MTT</td><td>0.625, 1.25, 2.5,<br>5, 10, 20</td><td>no info</td><td>IC<sub>50</sub> 3.1 µM</td><td>Zhao et al. (2019)</td></tr><tr><td>ALP</td><td>Brain</td><td>SF-268</td><td>SRB</td><td>No info</td><td>no info</td><td>IC<sub>50</sub> 6.57 µM</td><td>Wang et al. (2017)</td></tr><tr><td>ALS</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ALS</td><td>Colon</td><td>HCT116</td><td>SRB</td><td>No info</td><td>72</td><td>IC<sub>50</sub> 28.9 µM</td><td>Xiao et al. (2014)</td></tr><tr><td>ALS</td><td>Skin</td><td>HaCat</td><td>MTT</td><td>5, 10, 20, 40</td><td>24</td><td>effect > 40 µM</td><td>Dong et al. (2021)</td></tr><tr><td>ALS</td><td>Brain</td><td>BV2</td><td>MTT</td><td>0–100</td><td>24</td><td>no effect</td><td>Kumar et al. (2019)</td></tr><tr><td>ALT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ALT</td><td>Colon</td><td>HCT116</td><td>SRB</td><td>No info</td><td>72</td><td>IC<sub>50</sub> 3.13 µM</td><td>Xiao et al. (2014)</td></tr><tr><td>ALT</td><td>Skin</td><td>HaCat</td><td>MTT</td><td>10, 20, 40, 80</td><td>24</td><td>No effect</td><td>Dong et al. (2021)</td></tr><tr><td>AME</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>AME</td><td>Colon</td><td>Caco-2 undif-<br>ferentiated</td><td>MTT</td><td>3.125–100</td><td>24, 48</td><td>↓ viability (48 h),<br>no IC<sub>50</sub></td><td>Fernández-Blanco<br>et al. (2016a)</td></tr><tr><td>AME</td><td>Colon</td><td>Caco-2 undif-<br>ferentiated</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> up to<br>50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AME</td><td>Colon</td><td>Caco-2 undif-<br>ferentiated</td><td>FC</td><td>0.4, 0.9, 1.8,<br>3.7, 18.4,<br>36.7, 73.5,<br>220.4, 440.8</td><td>48</td><td>EC<sub>50</sub> 56.5 µM)</td><td>den Hollander<br>et al. (2022)</td></tr><tr><td>AME</td><td>Colon</td><td>HT29</td><td>LDH</td><td>0–50</td><td>1</td><td>No effect (data not<br>shown)</td><td>Fehr et al. (2009)</td></tr><tr><td>AME</td><td>Colon</td><td>HCT116</td><td>FDA</td><td>10, 25, 50,<br>100, 200</td><td>24</td><td>IC<sub>50</sub> 120 µM</td><td>Bensassi et al. (2011)</td></tr><tr><td>AME</td><td>Lung</td><td>V79</td><td>CC</td><td>10, 20, 30, 40</td><td>24</td><td>Accumulation in G2/M<br>phase > 10 µM</td><td>Fleck et al. (2012)</td></tr><tr><td>AME</td><td>Liver</td><td>HepG2</td><td>FC</td><td>0.4, 0.9, 1.8,<br>3.7, 18.4,<br>36.7, 73.5,<br>220.4, 440.8</td><td>24</td><td>EC<sub>50</sub> 18.6 µM</td><td>den Hollander<br>et al. (2022)</td></tr></tbody></table>

<figure><img src="image_6.png" /></figure>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>sToxin</th><th>Cell type</th><th>Cell model</th><th>Assay</th><th>Dose (µM)</th><th>Exposure time (h)</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>AME</td><td>Liver</td><td>HepaRG</td><td>MTT</td><td>0, 0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>No effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>AME</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>0, 0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>Dose-dependent effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>AME</td><td>Liver</td><td>HepG2</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> up to 50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AME</td><td>Liver</td><td>HepG2</td><td>AB</td><td>45.9, 91.8, 183.7, 367.3</td><td>No info</td><td>EC<sub>50</sub> 36 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>AME</td><td>Kidney</td><td>HEK 293 T</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> up to 50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AME</td><td>Skin</td><td>A431</td><td>LDH</td><td>0–50</td><td>1</td><td>No effect (data not shown)</td><td>Fehr et al. (2009)</td></tr><tr><td>AME</td><td>Ovary</td><td>PGC</td><td>MTT</td><td>0.8, 1.6, 3.2, 6.4, 12.8</td><td>24</td><td>Dose-dependent effect</td><td>Tiemann et al. (2009)</td></tr><tr><td>AME</td><td>Cervix</td><td>HeLa</td><td>AB</td><td>45.9, 91.8, 183.7, 367.3</td><td>No info</td><td>No effect</td><td>Mahmoud et al. (2022)</td></tr><tr><td>ATX-I</td><td>Liver</td><td>HepG2</td><td>AB</td><td>35.5, 71.0, 56.8, 283.8</td><td>No info</td><td>EC<sub>50</sub> 96.5 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>ATX-I</td><td>Mammary</td><td>MCF7</td><td>CTB</td><td>0.01, 0.1, 0.5, 1, 2.5, 5</td><td>24</td><td>No significant cytotoxicity</td><td>Hohenbichler et al. (2020)</td></tr><tr><td>ATX-II</td><td>Colon</td><td>HT29</td><td>WST-1</td><td>0.1, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25</td><td>24</td><td>EC<sub>50</sub> 16.5 µM</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>ATX-II</td><td>Colon</td><td>HCEC-1CT</td><td>WST-1</td><td>0.1, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25</td><td>24</td><td>EC<sub>50</sub> 6.9 µM</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>ATX-II</td><td>Colon</td><td>HT29</td><td>SRB</td><td>0.01, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 25, 50</td><td>24</td><td>dose-dependent ↓ > 0.2 µM</td><td>Tiessen et al. (2013a)</td></tr><tr><td>ATX-II</td><td>Colon</td><td>HT29</td><td>SRB</td><td>0.01, 0.05, 0.1, 0.2, 0.5, 1, 10</td><td>24, 72</td><td>Dose-dependent effect IC<sub>50</sub> 0.8 µM</td><td>Schwarz et al. (2012)</td></tr><tr><td>ATX-II</td><td>Lung</td><td>A549</td><td>AB</td><td>no info</td><td>72</td><td>EC<sub>50</sub> 1.15 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>ATX-II</td><td>Lung</td><td>V79</td><td>CC</td><td>0.1, 0.25, 0.5, 0.75</td><td>24</td><td>No effect</td><td>Fleck et al. (2012)</td></tr><tr><td>ATX-II</td><td>Liver</td><td>HepG2</td><td>AB</td><td>35.7, 71.4, 57.1, 285.5</td><td>No info</td><td>EC<sub>50</sub> 97.1 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>ATX-II</td><td>Liver</td><td>HepG2</td><td>WST-1</td><td>0.1, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25</td><td>24</td><td>EC<sub>50</sub> 7.3 µM</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>ATX-II</td><td>Prostate</td><td>PC3</td><td>AB</td><td>no info</td><td>72</td><td>EC<sub>50</sub> 0.33 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>STTX-III</td><td>Lung</td><td>V79</td><td>CC</td><td>0.1, 0.25, 0.5, 0.75</td><td>24</td><td>↓ cell number > 0.5 µM, ↓ plating efficiency > 0.25 µM</td><td>Fleck et al. (2012)</td></tr><tr><td>TeA</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>FC</td><td>0.5, 1.3, 2.5, 5.1, 25.4, 50.7, 101.4, 304.2, 608.4</td><td>48</td><td>EC<sub>50</sub> 356 µM</td><td>den Hollander et al. (2022)</td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>sToxin</th><th>Cell type</th><th>Cell model</th><th>Assay</th><th>Dose (µM)</th><th>Exposure time (h)</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>AME</td><td>Liver</td><td>HepaRG</td><td>MTT</td><td>0, 0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>No effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>AME</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>0, 0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>Dose-dependent effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>AME</td><td>Liver</td><td>HepG2</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> up to 50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AME</td><td>Liver</td><td>HepG2</td><td>AB</td><td>45.9, 91.8, 183.7, 367.3</td><td>No info</td><td>EC<sub>50</sub> 36 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>AME</td><td>Kidney</td><td>HEK 293 T</td><td>AB</td><td>0–0.05</td><td>72</td><td>No IC<sub>50</sub> up to 50 nM<sup>a</sup></td><td>Tran et al. (2020)</td></tr><tr><td>AME</td><td>Skin</td><td>A431</td><td>LDH</td><td>0–50</td><td>1</td><td>No effect (data not shown)</td><td>Fehr et al. (2009)</td></tr><tr><td>AME</td><td>Ovary</td><td>PGC</td><td>MTT</td><td>0.8, 1.6, 3.2, 6.4, 12.8</td><td>24</td><td>Dose-dependent effect</td><td>Tiemann et al. (2009)</td></tr><tr><td>AME</td><td>Cervix</td><td>HeLa</td><td>AB</td><td>45.9, 91.8, 183.7, 367.3</td><td>No info</td><td>No effect</td><td>Mahmoud et al. (2022)</td></tr><tr><td>ATX-I</td><td>Liver</td><td>HepG2</td><td>AB</td><td>35.5, 71.0, 56.8, 283.8</td><td>No info</td><td>EC<sub>50</sub> 96.5 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>ATX-I</td><td>Mammary</td><td>MCF7</td><td>CTB</td><td>0.01, 0.1, 0.5, 1, 2.5, 5</td><td>24</td><td>No significant cytotoxicity</td><td>Hohenbichler et al. (2020)</td></tr><tr><td>ATX-II</td><td>Colon</td><td>HT29</td><td>WST-1</td><td>0.1, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25</td><td>24</td><td>EC<sub>50</sub> 16.5 µM</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>ATX-II</td><td>Colon</td><td>HCEC-1CT</td><td>WST-1</td><td>0.1, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25</td><td>24</td><td>EC<sub>50</sub> 6.9 µM</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>ATX-II</td><td>Colon</td><td>HT29</td><td>SRB</td><td>0.01, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 25, 50</td><td>24</td><td>dose-dependent ↓ > 0.2 µM</td><td>Tiessen et al. (2013a)</td></tr><tr><td>ATX-II</td><td>Colon</td><td>HT29</td><td>SRB</td><td>0.01, 0.05, 0.1, 0.2, 0.5, 1, 10</td><td>24, 72</td><td>Dose-dependent effect IC<sub>50</sub> 0.8 µM</td><td>Schwarz et al. (2012)</td></tr><tr><td>ATX-II</td><td>Lung</td><td>A549</td><td>AB</td><td>no info</td><td>72</td><td>EC<sub>50</sub> 1.15 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>ATX-II</td><td>Lung</td><td>V79</td><td>CC</td><td>0.1, 0.25, 0.5, 0.75</td><td>24</td><td>No effect</td><td>Fleck et al. (2012)</td></tr><tr><td>ATX-II</td><td>Liver</td><td>HepG2</td><td>AB</td><td>35.7, 71.4, 57.1, 285.5</td><td>No info</td><td>EC<sub>50</sub> 97.1 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>ATX-II</td><td>Liver</td><td>HepG2</td><td>WST-1</td><td>0.1, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25</td><td>24</td><td>EC<sub>50</sub> 7.3 µM</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>ATX-II</td><td>Prostate</td><td>PC3</td><td>AB</td><td>no info</td><td>72</td><td>EC<sub>50</sub> 0.33 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>STTX-III</td><td>Lung</td><td>V79</td><td>CC</td><td>0.1, 0.25, 0.5, 0.75</td><td>24</td><td>↓ cell number > 0.5 µM, ↓ plating efficiency > 0.25 µM</td><td>Fleck et al. (2012)</td></tr><tr><td>TeA</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>FC</td><td>0.5, 1.3, 2.5, 5.1, 25.4, 50.7, 101.4, 304.2, 608.4</td><td>48</td><td>EC<sub>50</sub> 356 µM</td><td>den Hollander et al. (2022)</td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>sToxin</th><th>Cell type</th><th>Cell model</th><th>Assay</th><th>Dose (µM)</th><th>Exposure time (h)</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>TeA</td><td>Liver</td><td>HepG2</td><td>FC</td><td>0.5, 1.3, 2.5, 5.1, 25.4, 50.7, 101.4, 304.2, 608.4</td><td>24</td><td>EC<sub>50</sub> 380.8 µM</td><td>den Hollander et al. (2022)</td></tr><tr><td>TeA</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>Dose-dependent effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>TeA</td><td>Liver</td><td>HepG2</td><td>AB</td><td>63.4, 126.8, 253.5, 507.0</td><td>No info</td><td>EC<sub>50</sub> 146 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>TeA</td><td>Liver</td><td>HepaRG</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>Dose-dependent effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>TeA</td><td>Cervix</td><td>HeLa</td><td>AB</td><td>63.4, 126.8, 253.5, 507.0</td><td>No info</td><td>EC<sub>50</sub> 109 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>TeA</td><td>Ovary</td><td>PGC</td><td>MTT</td><td>6.4, 12.8, 25, 50, 100</td><td>24</td><td>No effect</td><td>Tiemann et al. (2009)</td></tr><tr><td>TEN</td><td>Liver</td><td>HepaRG</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>No effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>TEN</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>No effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>AOH (combi with DON, UroA)</td><td>Colon</td><td>Caco-2 differentiated</td><td>NR</td><td>25</td><td>48</td><td>↓cell viability at 2.5 µM of deoxynivalenol + 25 µM AOH</td><td>Groestlinger et al. (2022)</td></tr><tr><td>Combination AOH, 3-ADON and 15-ADON</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>AOH: 3.2, 6.4, 12.8, 24</td><td>24, 48, 72</td><td>IC<sub>50</sub> values at all times assayed, from 0.8 to ≥ 25 µM in binary combinations; while in tertiary from 7.5-12 µM ↓ cell viability > 12.8 µM but IC<sub>50</sub> not reached</td><td>Juan-García et al. (2016)</td></tr><tr><td>AME: AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>MTT</td><td>3.125, 6.25, 12.5, 25, 50, 100</td><td>24, 48</td><td>Synergistic effect</td><td>Fernández-Blanco et al. (2016a)</td></tr><tr><td>ATX-II: AOH</td><td>Colon</td><td>HT29</td><td>WST-1</td><td>0.1, 0.5, 1, 2.5, 5, 10, 25</td><td>24</td><td>Combinatory effect (antagonism)</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>ATX-II: AOH</td><td>Colon</td><td>HCEC-1CT</td><td>WST-1</td><td>0.1, 0.5, 1, 2.5, 5, 10, 25</td><td>24</td><td>Combinatory effect (antagonism)</td><td>Vejdovszky et al. (2017)</td></tr></tbody></table>

Cell lines: **184A1** human mammary gland, **A431** human vulva carcinoma, **A549** human lung carcinoma, **BEAS-2B** human bronchial epithelial, **BV2** murine microglia, **Caco-2** human colon carcinoma, **CHO-K1** Chinese hamster ovary, **H295R** human adrenocortical carcinoma, **HaCaT** human keratinocytes, **HCEC-1CT** immortalized human colon epithelial, **HCT116** human colon carcinoma, **HEK 293T** human embryonal kidney, **HeLa** human cervical carcinoma, **HepG2** human liver carcinoma, **HepaRG** human liver carcinoma, **HT29** human colon carcinoma, **Ishikawa** human endometrial adenocarcinoma, **KYSE510** human esophageal squamous carcinoma, **LLC-PK1** pig kidney epithelial cells, **MCF-7** human breast adenocarcinoma, **MMV-Luc** RGA human mammary gland, **NCIH460** human lung carcinoma, **PC3** human prostatic small cell carcinoma, **PGC** porcine granulosa, **PNT1A** human primary prostate epithelial, **RAW264.7** mouse macrophage, **RGA** luciferase transfected human mammary gland, **SF-268** human astrocytoma, **THP1-Lucia™ NF-κBs** luciferase transfected human monocytic leukemia, **THP1** human monocytic leukemia, **TARM-Luc** RGA human mammary gland androgen and progestagen responsive, **TGRM-Luc** RGA human mammary gland glucocorticoid and progestagen responsive, **TM-Luc** RGA human mammary gland progestagen responsive, **V79** hamster lung fibroblasts

Assays **AB** Alamar Blue, **A/N** apoptosis/necrosis, **CC** Cell cycle, **CTB** CellTiter blue, **FC** flow cytometry, **FDA** fluorescein diacetate, **LDH** Lactate dehydrogenase, **MTT** 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide, **NR** Neutral Red, **PC** protein content assay, **SRB** sulforhodamine B, **TB** trypan blue, **WST-1** tetrazolium dye

<figure><img src="image_5.png" /></figure>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>sToxin</th><th>Cell type</th><th>Cell model</th><th>Assay</th><th>Dose (µM)</th><th>Exposure time (h)</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>TeA</td><td>Liver</td><td>HepG2</td><td>FC</td><td>0.5, 1.3, 2.5, 5.1, 25.4, 50.7, 101.4, 304.2, 608.4</td><td>24</td><td>EC<sub>50</sub> 380.8 µM</td><td>den Hollander et al. (2022)</td></tr><tr><td>TeA</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>Dose-dependent effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>TeA</td><td>Liver</td><td>HepG2</td><td>AB</td><td>63.4, 126.8, 253.5, 507.0</td><td>No info</td><td>EC<sub>50</sub> 146 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>TeA</td><td>Liver</td><td>HepaRG</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>Dose-dependent effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>TeA</td><td>Cervix</td><td>HeLa</td><td>AB</td><td>63.4, 126.8, 253.5, 507.0</td><td>No info</td><td>EC<sub>50</sub> 109 µM</td><td>Mahmoud et al. (2022)</td></tr><tr><td>TeA</td><td>Ovary</td><td>PGC</td><td>MTT</td><td>6.4, 12.8, 25, 50, 100</td><td>24</td><td>No effect</td><td>Tiemann et al. (2009)</td></tr><tr><td>TEN</td><td>Liver</td><td>HepaRG</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>No effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>TEN</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>0.01, 0.1, 1, 10, 50, 100</td><td>24</td><td>No effect</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>AOH (combi with DON, UroA)</td><td>Colon</td><td>Caco-2 differentiated</td><td>NR</td><td>25</td><td>48</td><td>↓cell viability at 2.5 µM of deoxynivalenol + 25 µM AOH</td><td>Groestlinger et al. (2022)</td></tr><tr><td>Combination AOH, 3-ADON and 15-ADON</td><td>Liver</td><td>HepG2</td><td>MTT</td><td>AOH: 3.2, 6.4, 12.8, 24</td><td>24, 48, 72</td><td>IC<sub>50</sub> values at all times assayed, from 0.8 to ≥ 25 µM in binary combinations; while in tertiary from 7.5-12 µM ↓ cell viability > 12.8 µM but IC<sub>50</sub> not reached</td><td>Juan-García et al. (2016)</td></tr><tr><td>AME: AOH</td><td>Colon</td><td>Caco-2 undifferentiated</td><td>MTT</td><td>3.125, 6.25, 12.5, 25, 50, 100</td><td>24, 48</td><td>Synergistic effect</td><td>Fernández-Blanco et al. (2016a)</td></tr><tr><td>ATX-II: AOH</td><td>Colon</td><td>HT29</td><td>WST-1</td><td>0.1, 0.5, 1, 2.5, 5, 10, 25</td><td>24</td><td>Combinatory effect (antagonism)</td><td>Vejdovszky et al. (2017)</td></tr><tr><td>ATX-II: AOH</td><td>Colon</td><td>HCEC-1CT</td><td>WST-1</td><td>0.1, 0.5, 1, 2.5, 5, 10, 25</td><td>24</td><td>Combinatory effect (antagonism)</td><td>Vejdovszky et al. (2017)</td></tr></tbody></table>

Cell lines: **184A1** human mammary gland, **A431** human vulva carcinoma, **A549** human lung carcinoma, **BEAS-2B** human bronchial epithelial, **BV2** murine microglia, **Caco-2** human colon carcinoma, **CHO-K1** Chinese hamster ovary, **H295R** human adrenocortical carcinoma, **HaCaT** human keratinocytes, **HCEC-1CT** immortalized human colon epithelial, **HCT116** human colon carcinoma, **HEK 293T** human embryonal kidney, **HeLa** human cervical carcinoma, **HepG2** human liver carcinoma, **HepaRG** human liver carcinoma, **HT29** human colon carcinoma, **Ishikawa** human endometrial adenocarcinoma, **KYSE510** human esophageal squamous carcinoma, **LLC-PK1** pig kidney epithelial cells, **MCF-7** human breast adenocarcinoma, **MMV-Luc** RGA human mammary gland, **NCIH460** human lung carcinoma, **PC3** human prostatic small cell carcinoma, **PGC** porcine granulosa, **PNT1A** human primary prostate epithelial, **RAW264.7** mouse macrophage, **RGA** luciferase transfected human mammary gland, **SF-268** human astrocytoma, **THP1-Lucia™ NF-κBs** luciferase transfected human monocytic leukemia, **THP1** human monocytic leukemia, **TARM-Luc** RGA human mammary gland androgen and progestagen responsive, **TGRM-Luc** RGA human mammary gland glucocorticoid and progestagen responsive, **TM-Luc** RGA human mammary gland progestagen responsive, **V79** hamster lung fibroblasts

Assays **AB** Alamar Blue, **A/N** apoptosis/necrosis, **CC** Cell cycle, **CTB** CellTiter blue, **FC** flow cytometry, **FDA** fluorescein diacetate, **LDH** Lactate dehydrogenase, **MTT** 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide, **NR** Neutral Red, **PC** protein content assay, **SRB** sulforhodamine B, **TB** trypan blue, **WST-1** tetrazolium dye
Caco-2 cells nor in non-stimulated cells after incubation with 0.02–40 µM AOH for 5 h and 20 h (Schmutz et al. 2019). In vitro cytotoxicity studies in Caco-2 cells resulted in EC₅₀ values of 19 µg/mL, 6–23 µg/mL, and 60–90 µg/mL for, respectively, AOH, AME and TeA (corresponding to 73 µM, 22–84 µM and 304–456 µM). Incubation of undifferentiated Caco-2 cells with 60 µM AOH for 24 h resulted in a significant decrease in the intracellular glutathione level (Chiesi et al. 2015; Fernández-Blanco et al. 2015).

In the human colon carcinoma cell line HCT116, a concentration-dependent reduction of cell viability at 10–200 µM AOH was observed after 24 h using the fluorescent probe fluorescein diacetate (FDA), with IC₅₀ determined at 65 µM (Bensassi et al. 2011, 2012). In HT29 cells, short-time incubation (1 h) with AOH did not significantly affect the leakage of LDH (Fehr et al. 2009), whereas prolonged incubation (24 h) induced cytotoxic effects, starting at 25 and 50 µM in the WST-1 and SRB assay, respectively (Aichinger et al. 2017). In addition, a growth inhibitory effect of AOH in HT29 cells was associated with concentration-dependent ROS formation (Pahlke et al. 2016).

Upon 48-h incubation with AOH, an onset of cytotoxicity was observed at concentrations ≥ 5 µM in Ishikawa cells (Aichinger et al. 2020b). In HeLa cells exposed to 0–250 µM AOH for 24 h (in the absence of FBS to avoid interference), a concentration-dependent decrease in ATP levels was shown with a significant decrease noticed at 10 µM, and an IC₅₀ of 33.6 µM (Balázs et al. 2021). Using the Alamar blue (AB) and MTT assays in the human adrenocortical carcinoma cell line H295R, exposed for 48 h to 0.1–1000 ng/mL, AOH had no effect. AOH reduced cell viability above 5000 ng/mL in three out the four human mammary gland cell lines (RGA) exposed from 50 to 10,000 ng/mL (Frizzell et al. 2013). AOH significantly affected the cell viability of prostate epithelial cells (PNT1A cells) using the AB assay, leading to about 40% reduction at 10 µM. In addition, the AOH exposure led to a cell cycle arrest in the G2/M phase (Kowalska et al. 2021b). In mammary 184A1 cells, AOH significantly affected the cell viability in a time- and dose-dependent manner (Kowalska et al. 2021a).

In HepG2, IC₅₀ of 96 µM (24 h), 90 µM (48 h), and 65 µM (72 h) were determined for AOH by Juan-Garcia et al. 2015. When the WST-1 assay was used, HepG2 showed higher sensitivity to AOH compared to HT29 and HCEC-1CT. A comparable sensitivity of non-tumorigenic intestinal cell HCEC-1CT toward AOH and ATX-II was observed. HT29 were the least sensitive to both mycotoxins (Vejdovszky et al. 2017). In a study using the MTT assay, 24-h incubation to AOH induced significant cytotoxic effects in HepG2 and HepaRG at the highest test concentration of 100 µM without substantial differences between both cell types (Hessel-Pras et al. 2019).

In THP1 monocytes, metabolic activity measured by the AB assay was decreased by 25% at 7.5 µM AOH, and more at higher concentrations due to a G2 phase arrest in the cell cycle. Only low levels of necrosis and apoptosis were found (Solhaug et al. 2016a). Furthermore, the AB assay was used in THP1-derived macrophages and THP1-Lucia™ NF-κB cells (differentiated). After exposure for 5 and 20 h to AOH concentrations ranging from 0.02 to 20 µM, cytotoxic effects were only observed in both cell lines at 20 µM (20 h) after stimulation with lipopolysaccharide (LPS) (no cytotoxicity in non-stimulated cells) (Kollarova et al. 2018). In the murine macrophage cell line RAW264.7, AOH reduced the viability after 24-h exposure, with EC₅₀ of 49.65 µM and 78.01 using the AB and the neutral red assay, respectively. Apoptosis and necrosis were only found at a higher concentration (60 µM) or a more prolonged exposure (48 h) (Solhaug et al. 2012). In primary blood-derived human macrophages and primary peritoneal murine macrophages, exposure to AOH up to 60 µM for 24–48 h did not induce cell death (Solhaug et al. 2015). When pig granulosa cells (ovary cells) were exposed for 24 h to AOH, they showed a reduced viability at ≥ 1.6 µM AOH in the MTT assay (Tiemann et al. 2009). In Ishikawa and V79 cells, flow cytometry indicated that AOH exposure reduced cell proliferation and increased the number of cells in the G2/M phase at 5 and 10 µM (Lehmann et al. 2006).

Oxidative stress is likely to contribute to the cytotoxicity of AOH. In Caco-2 cells, AOH induced oxidative stress by ROS and LPO expression (Fernández-Blanco et al. 2016b). Pahlke et al. studied the impact of AOH and AME on cytochrome P450 (CYP)1A1 expression, ROS production and cytotoxicity in human HT29 cells (Pahlke et al. 2016). A growth inhibitory effect of AOH and AME in HT29 correlated with dose-dependent ROS formation. In Chinese hamster V79 lung fibroblasts and in mouse lymphoma LY5178Ytk + / - cells (MLC), a concentration-dependent reduction of viable cells was observed with, respectively, up to 30 µM AME and 20 µM AOH (Brugger et al. 2006). Also, in RAW 264.7 mouse macrophages AOH (30 µM) exposure led to the production of ROS, but it was not associated with cell cycle arrest (Solhaug et al. 2012).

For AME, a broad range of cytotoxic effects in the low micromolar range has been reported, whereby the limited solubility of the compound might play a certain role. Comparably to AOH, AME did not significantly affect LDH leakage after 1-h exposure of HT29 cells (Fehr et al. 2009). After 24 h of HT29 cells at concentrations above 25 µM, an onset of toxicity was observed in the SRB assay (Tiessen et al. 2013a). In HepG2 cells, AME > 10 µM resulted in significant cytotoxic effects after 24-h incubation as measured in the MTT assay and HepG2 cells were clearly more sensitive to AME than HepaRG cells (Hessel-Pras et al. 2019). PGC exposed to AME for 24 h showed a reduced viability
Caco-2 cells nor in non-stimulated cells after incubation with 0.02–40 µM AOH for 5 h and 20 h (Schmutz et al. 2019). In vitro cytotoxicity studies in Caco-2 cells resulted in EC₅₀ values of 19 µg/mL, 6–23 µg/mL, and 60–90 µg/mL for, respectively, AOH, AME and TeA (corresponding to 73 µM, 22–84 µM and 304–456 µM). Incubation of undifferentiated Caco-2 cells with 60 µM AOH for 24 h resulted in a significant decrease in the intracellular glutathione level (Chiesi et al. 2015; Fernández-Blanco et al. 2015).

In the human colon carcinoma cell line HCT116, a concentration-dependent reduction of cell viability at 10–200 µM AOH was observed after 24 h using the fluorescent probe fluorescein diacetate (FDA), with IC₅₀ determined at 65 µM (Bensassi et al. 2011, 2012). In HT29 cells, short-time incubation (1 h) with AOH did not significantly affect the leakage of LDH (Fehr et al. 2009), whereas prolonged incubation (24 h) induced cytotoxic effects, starting at 25 and 50 µM in the WST-1 and SRB assay, respectively (Aichinger et al. 2017). In addition, a growth inhibitory effect of AOH in HT29 cells was associated with concentration-dependent ROS formation (Pahlke et al. 2016).

Upon 48-h incubation with AOH, an onset of cytotoxicity was observed at concentrations ≥ 5 µM in Ishikawa cells (Aichinger et al. 2020b). In HeLa cells exposed to 0–250 µM AOH for 24 h (in the absence of FBS to avoid interference), a concentration-dependent decrease in ATP levels was shown with a significant decrease noticed at 10 µM, and an IC₅₀ of 33.6 µM (Balázs et al. 2021). Using the Alamar blue (AB) and MTT assays in the human adrenocortical carcinoma cell line H295R, exposed for 48 h to 0.1–1000 ng/mL, AOH had no effect. AOH reduced cell viability above 5000 ng/mL in three out the four human mammary gland cell lines (RGA) exposed from 50 to 10,000 ng/mL (Frizzell et al. 2013). AOH significantly affected the cell viability of prostate epithelial cells (PNT1A cells) using the AB assay, leading to about 40% reduction at 10 µM. In addition, the AOH exposure led to a cell cycle arrest in the G2/M phase (Kowalska et al. 2021b). In mammary 184A1 cells, AOH significantly affected the cell viability in a time- and dose-dependent manner (Kowalska et al. 2021a).

In HepG2, IC₅₀ of 96 µM (24 h), 90 µM (48 h), and 65 µM (72 h) were determined for AOH by Juan-Garcia et al. 2015. When the WST-1 assay was used, HepG2 showed higher sensitivity to AOH compared to HT29 and HCEC-1CT. A comparable sensitivity of non-tumorigenic intestinal cell HCEC-1CT toward AOH and ATX-II was observed. HT29 were the least sensitive to both mycotoxins (Vejdovszky et al. 2017). In a study using the MTT assay, 24-h incubation to AOH induced significant cytotoxic effects in HepG2 and HepaRG at the highest test concentration of 100 µM without substantial differences between both cell types (Hessel-Pras et al. 2019).

In THP1 monocytes, metabolic activity measured by the AB assay was decreased by 25% at 7.5 µM AOH, and more at higher concentrations due to a G2 phase arrest in the cell cycle. Only low levels of necrosis and apoptosis were found (Solhaug et al. 2016a). Furthermore, the AB assay was used in THP1-derived macrophages and THP1-Lucia™ NF-κB cells (differentiated). After exposure for 5 and 20 h to AOH concentrations ranging from 0.02 to 20 µM, cytotoxic effects were only observed in both cell lines at 20 µM (20 h) after stimulation with lipopolysaccharide (LPS) (no cytotoxicity in non-stimulated cells) (Kollarova et al. 2018). In the murine macrophage cell line RAW264.7, AOH reduced the viability after 24-h exposure, with EC₅₀ of 49.65 µM and 78.01 using the AB and the neutral red assay, respectively. Apoptosis and necrosis were only found at a higher concentration (60 µM) or a more prolonged exposure (48 h) (Solhaug et al. 2012). In primary blood-derived human macrophages and primary peritoneal murine macrophages, exposure to AOH up to 60 µM for 24–48 h did not induce cell death (Solhaug et al. 2015). When pig granulosa cells (ovary cells) were exposed for 24 h to AOH, they showed a reduced viability at ≥ 1.6 µM AOH in the MTT assay (Tiemann et al. 2009). In Ishikawa and V79 cells, flow cytometry indicated that AOH exposure reduced cell proliferation and increased the number of cells in the G2/M phase at 5 and 10 µM (Lehmann et al. 2006).

Oxidative stress is likely to contribute to the cytotoxicity of AOH. In Caco-2 cells, AOH induced oxidative stress by ROS and LPO expression (Fernández-Blanco et al. 2016b). Pahlke et al. studied the impact of AOH and AME on cytochrome P450 (CYP)1A1 expression, ROS production and cytotoxicity in human HT29 cells (Pahlke et al. 2016). A growth inhibitory effect of AOH and AME in HT29 correlated with dose-dependent ROS formation. In Chinese hamster V79 lung fibroblasts and in mouse lymphoma LY5178Ytk + / - cells (MLC), a concentration-dependent reduction of viable cells was observed with, respectively, up to 30 µM AME and 20 µM AOH (Brugger et al. 2006). Also, in RAW 264.7 mouse macrophages AOH (30 µM) exposure led to the production of ROS, but it was not associated with cell cycle arrest (Solhaug et al. 2012).

For AME, a broad range of cytotoxic effects in the low micromolar range has been reported, whereby the limited solubility of the compound might play a certain role. Comparably to AOH, AME did not significantly affect LDH leakage after 1-h exposure of HT29 cells (Fehr et al. 2009). After 24 h of HT29 cells at concentrations above 25 µM, an onset of toxicity was observed in the SRB assay (Tiessen et al. 2013a). In HepG2 cells, AME > 10 µM resulted in significant cytotoxic effects after 24-h incubation as measured in the MTT assay and HepG2 cells were clearly more sensitive to AME than HepaRG cells (Hessel-Pras et al. 2019). PGC exposed to AME for 24 h showed a reduced viability
at concentrations ≥ 1.6 µM in the MTT assay (Tiemann et al. 2009).

ALS induced cytotoxic effects as detected in the SRB assay after 72-h exposure in HCT116 cells ($IC_{50}$=28.9 µM) (Xiao et al. 2014). In HaCaT cells, cytotoxic effects were detected at concentrations > 40 µM after 24-h incubation (Dong et al. 2021). In contrast, no toxicity was detected with up to 100 µM ALS in BV2 microglial cells (LPS-stimulated for 24 h) using the MTT assay (Kumar et al. 2019).

ALT did not impact cell viability (MTT assay) in the HaCaT keratinocyte cell line below 80 µM after 24 h (Dong et al. 2021). In contrast, cytotoxicity was reported in HCT116 cells after 72 h exposure to ALT (SRB assay), reaching an $IC_{50}$ of 3.13 µM (Xiao et al. 2014).

ALP exposure led to a determinable $IC_{50}$ in all cell models used. In MCF7, HepG2, NCIH460, and SF-268 cells, using the SRB assay, $IC_{50}$ values ranged from 3.73 to 6.57 µM (Wang et al. 2017). Similarly, ALP cytotoxicity in A549, HCT116, and HeLa cells was comparable with $IC_{50}$ values of 2.6, 2.4, and 3.1 µM, respectively (Zhao et al. 2019).

ATX-II did not decrease viability in intestinal HT29 cells (Trypan Blue exclusion assay) after 1 and 24 h exposure to 0.01–1 µM, while a concentration-dependent decrease of cell proliferation (SRB assay) was detected at ≥ 0.05 µM ATX-II after 24 h and 72 h, with an $IC_{50}$ of 0.8 µM after 72 h (Schwarz et al. 2012). ATX-II was cytotoxic (WST-1 assay) in HepG2, HT29 and HCEC-1CT cells. As compared to AOH, the sensitivity of HCEC-1CT cells to ATX-II was similar, while it was less for HepG2 cells. HT29 cells showed the least sensitivity to both mycotoxins (Vejdovszky et al. 2017).

There is only one cytotoxicity study on STTX-III, reporting a reduction in V79 cell numbers at concentrations > 0.5 µM, and a reduction in RPE at concentrations > 0.25 µM (Fleck et al. 2016).

In vitro cytotoxicity studies in undifferentiated Caco-2 cells resulted in $EC_{50}$ values between 60 and 90 µM TeA (den Hollander et al. 2022). In Hela cells, the $EC_{50}$ was 146 µM (Mahmoud et al. 2022). After 24 h, TeA induced a concentration-dependent cytotoxicity in HepG2 cells (significant at 100 µM), which was more pronounced than in HepaRG cells (Hessel-Pras et al. 2019). In contrast, cell viability (MTT assay) was not decreased in pig granulosa cells (ovary cells) exposed for 24 h to TeA up to 100 µM (Tiemann et al. 2009).

## Mixtures

The cytotoxicity of individual *Alternaria* toxins depends greatly on the cell line used, and on the toxin tested. As mycotoxins occur usually in mixtures in food and feed commodities, the situation gets even more complex, because

effects of toxin mixtures must be determined. For this purpose, combinatory studies are needed to decipher if additivity, antagonism or synergism needs to be considered in health risk assessment.

Cell viability was more reduced when human intestinal cells (HCT116) were exposed to AOH and AME (1:1 ratio) together than by individual exposure (Bensassi et al. 2015). Both, separately and mixed, AOH and the *Fusarium* toxin deoxynivalenol (DON) significantly increased the transepithelial electrical resistance (TEER) in Caco-2 cells mostly in an additive manner. The combination also enhanced the expression of the tight junction (TJ) protein ZO-1, thus potentially affecting the permeability of the gastrointestinal barrier in differentiated Caco-2 cells (Groestlinger et al. 2022). Cytotoxicity measurements of 1:10 or 1:1 ATX-II to AOH revealed additive effects in HepG2, HT29 and HCEC-1CT cells (Vejdovszky et al. 2017). Whereas AOH alone reduced cellular proliferation of undifferentiated Caco-2 cells in a concentration-dependent manner, the combination of AOH with tyrosol had a protective effect (Chiesi et al. 2015). Binary combinations of especially AME and TeA (1:3 ratio), but also AOH and AME (1:1 ratio), significantly increased the cytotoxicity as compared to the single compounds (den Hollander et al. 2022). Treatment with binary and ternary mixtures of AOH, AME and TeA in a 1:1:3 ratio showed a concentration-dependent decrease in undifferentiated Caco-2 cell viability (den Hollander et al. 2022), but mathematical models to dissect between additive, synergistic or antagonistic effects were not applied. In a study investigating the combinatory estrogenic effects of bisphenol A (BPA) with AOH and the *Fusarium* mycoestrogen zearalenone (ZEN) in Ishikawa cells (endometrial adenocarcinoma cells), cytotoxicity was monitored by neutral red assay (Aichinger et al. 2020b). BPA and ZEN were applied in non-cytotoxic concentrations to allow estrogenic stimuli and combinatory cytotoxic effects were not observed. In a 1:1 mixture of AOH and BPA (up to 10 µM), cytotoxicity was clearly dominated by AOH (Aichinger et al. 2020c).

## Summary of cytotoxicity studies

The multiple assays that have addressed the in vitro cytotoxicity of *Alternaria* mycotoxins demonstrated notable differences in potency and elicited effect, showing considerable toxicity for some of the toxins. The majority of the studies were performed using AOH: 36 out of 48 revealed cytotoxic effects, with a broad range of effective concentrations, reporting $EC_{50}$ values between 2.69 and 108.4 µM (Table 1). Of the 15 studies focused on AME, 8 did not show cytotoxic effects, and the others reported $IC_{50}$ of 9.8 or 18.6 µM in liver cells and 56.5 or 120 µM in intestinal cells, suggesting that the liver might represent a more susceptible organ. However, partial discrepancies are noted between studies

<figure><img src="image_6.png" /></figure>
at concentrations ≥ 1.6 µM in the MTT assay (Tiemann et al. 2009).

ALS induced cytotoxic effects as detected in the SRB assay after 72-h exposure in HCT116 cells ($IC_{50}$=28.9 µM) (Xiao et al. 2014). In HaCaT cells, cytotoxic effects were detected at concentrations > 40 µM after 24-h incubation (Dong et al. 2021). In contrast, no toxicity was detected with up to 100 µM ALS in BV2 microglial cells (LPS-stimulated for 24 h) using the MTT assay (Kumar et al. 2019).

ALT did not impact cell viability (MTT assay) in the HaCaT keratinocyte cell line below 80 µM after 24 h (Dong et al. 2021). In contrast, cytotoxicity was reported in HCT116 cells after 72 h exposure to ALT (SRB assay), reaching an $IC_{50}$ of 3.13 µM (Xiao et al. 2014).

ALP exposure led to a determinable $IC_{50}$ in all cell models used. In MCF7, HepG2, NCIH460, and SF-268 cells, using the SRB assay, $IC_{50}$ values ranged from 3.73 to 6.57 µM (Wang et al. 2017). Similarly, ALP cytotoxicity in A549, HCT116, and HeLa cells was comparable with $IC_{50}$ values of 2.6, 2.4, and 3.1 µM, respectively (Zhao et al. 2019).

ATX-II did not decrease viability in intestinal HT29 cells (Trypan Blue exclusion assay) after 1 and 24 h exposure to 0.01–1 µM, while a concentration-dependent decrease of cell proliferation (SRB assay) was detected at ≥ 0.05 µM ATX-II after 24 h and 72 h, with an $IC_{50}$ of 0.8 µM after 72 h (Schwarz et al. 2012). ATX-II was cytotoxic (WST-1 assay) in HepG2, HT29 and HCEC-1CT cells. As compared to AOH, the sensitivity of HCEC-1CT cells to ATX-II was similar, while it was less for HepG2 cells. HT29 cells showed the least sensitivity to both mycotoxins (Vejdovszky et al. 2017).

There is only one cytotoxicity study on STTX-III, reporting a reduction in V79 cell numbers at concentrations > 0.5 µM, and a reduction in RPE at concentrations > 0.25 µM (Fleck et al. 2016).

In vitro cytotoxicity studies in undifferentiated Caco-2 cells resulted in $EC_{50}$ values between 60 and 90 µM TeA (den Hollander et al. 2022). In Hela cells, the $EC_{50}$ was 146 µM (Mahmoud et al. 2022). After 24 h, TeA induced a concentration-dependent cytotoxicity in HepG2 cells (significant at 100 µM), which was more pronounced than in HepaRG cells (Hessel-Pras et al. 2019). In contrast, cell viability (MTT assay) was not decreased in pig granulosa cells (ovary cells) exposed for 24 h to TeA up to 100 µM (Tiemann et al. 2009).

## Mixtures

The cytotoxicity of individual *Alternaria* toxins depends greatly on the cell line used, and on the toxin tested. As mycotoxins occur usually in mixtures in food and feed commodities, the situation gets even more complex, because

effects of toxin mixtures must be determined. For this purpose, combinatory studies are needed to decipher if additivity, antagonism or synergism needs to be considered in health risk assessment.

Cell viability was more reduced when human intestinal cells (HCT116) were exposed to AOH and AME (1:1 ratio) together than by individual exposure (Bensassi et al. 2015). Both, separately and mixed, AOH and the *Fusarium* toxin deoxynivalenol (DON) significantly increased the transepithelial electrical resistance (TEER) in Caco-2 cells mostly in an additive manner. The combination also enhanced the expression of the tight junction (TJ) protein ZO-1, thus potentially affecting the permeability of the gastrointestinal barrier in differentiated Caco-2 cells (Groestlinger et al. 2022). Cytotoxicity measurements of 1:10 or 1:1 ATX-II to AOH revealed additive effects in HepG2, HT29 and HCEC-1CT cells (Vejdovszky et al. 2017). Whereas AOH alone reduced cellular proliferation of undifferentiated Caco-2 cells in a concentration-dependent manner, the combination of AOH with tyrosol had a protective effect (Chiesi et al. 2015). Binary combinations of especially AME and TeA (1:3 ratio), but also AOH and AME (1:1 ratio), significantly increased the cytotoxicity as compared to the single compounds (den Hollander et al. 2022). Treatment with binary and ternary mixtures of AOH, AME and TeA in a 1:1:3 ratio showed a concentration-dependent decrease in undifferentiated Caco-2 cell viability (den Hollander et al. 2022), but mathematical models to dissect between additive, synergistic or antagonistic effects were not applied. In a study investigating the combinatory estrogenic effects of bisphenol A (BPA) with AOH and the *Fusarium* mycoestrogen zearalenone (ZEN) in Ishikawa cells (endometrial adenocarcinoma cells), cytotoxicity was monitored by neutral red assay (Aichinger et al. 2020b). BPA and ZEN were applied in non-cytotoxic concentrations to allow estrogenic stimuli and combinatory cytotoxic effects were not observed. In a 1:1 mixture of AOH and BPA (up to 10 µM), cytotoxicity was clearly dominated by AOH (Aichinger et al. 2020c).

## Summary of cytotoxicity studies

The multiple assays that have addressed the in vitro cytotoxicity of *Alternaria* mycotoxins demonstrated notable differences in potency and elicited effect, showing considerable toxicity for some of the toxins. The majority of the studies were performed using AOH: 36 out of 48 revealed cytotoxic effects, with a broad range of effective concentrations, reporting $EC_{50}$ values between 2.69 and 108.4 µM (Table 1). Of the 15 studies focused on AME, 8 did not show cytotoxic effects, and the others reported $IC_{50}$ of 9.8 or 18.6 µM in liver cells and 56.5 or 120 µM in intestinal cells, suggesting that the liver might represent a more susceptible organ. However, partial discrepancies are noted between studies
using the same cell model and comparable dose range. In direct comparison, proliferating cells (HepG2) appear to be more affected by AME than differentiated liver cells (Hep-aRG), raising the question of whether tissue origin or proliferation/differentiation status represents the most important susceptibility factor.

Contradictory results were reported for ALT cytotoxicity in the keratinocyte cell line HaCaT, as well as in HCT116 cells (Table 1). ALS had an overall much higher IC₅₀, between 28.9 and 40 µM. For TeA, IC₅₀ were reported in the range of 70.2–146 µM in most studies. TEN did not show cytotoxic effects in one study in liver cells at levels < 100 µM.

Cytotoxicity data for perylene quinones are still limited. ALP, an analog without epoxide moiety, showed substantial cytotoxic properties in the low micromolar range (IC₅₀ ranging from 2.6 to 6.57 µM). For ATX-I, differing from ALP only by one double bond, the two available reports provided contradictory results. ATX-II represents a perylene quinone with a reactive epoxide moiety. The available studies showed IC₅₀ values ranging from 0.4 to 16.5 µM. The only study using the epoxide-bearing STTX-III showed cytotoxicity above 0.25 µM. Taken together, the cytotoxic properties of ATX-I remain to be clarified, but within the class of perylene quinones cytotoxicity appears not to be limited to the epoxide-bearing analogs since cytotoxic effects have been reported also for ALP. Nevertheless, the limited data available indicate that from the compounds tested so far, ATX-II and STTX-III represent the *Alternaria* toxins with the highest cytotoxic potential. For all studies on *Alternaria* perylene quinones, structural characterization and purity of the compounds are critical factors for the interpretation of the results. So far, these perylene quinones are not accessible via chemical synthesis but need to be isolated from respective fungal cultures. Thereby, the reactivity of the epoxide-bearing analogs (e.g., ATX-II, ATX-III, STTX-III) might lead to unexpected loss of intact test compound, which could underestimate toxicity. On the other hand, traces of these highly toxic epoxide-bearing compounds in preparations of non-epoxide analogs might generate misleading results on apparent toxicity, thus probably overestimating the toxic potential of some isolated analogs.

In summary, most evidence suggests that several *Alternaria* toxins have a cytotoxic potential, some already at nanomolar concentrations. However, some contradictions in the in vitro studies reported indicate that further testing using current guidelines/guidance is needed (e.g., OECD guidelines). Attention should be directed toward the characterization of the toxins present in a cytotoxicity assay, since co-contamination with more than one toxin may introduce bias in the outcome and can cause discrepancies between the studies reported. Most of the test compounds used in the above-cited studies originated from natural sources. Thus,

differences in purity can strongly affect the test results. The combined effects of *Alternaria* toxins that frequently co-occur have been poorly studied, but first results already pointed at the existence of increased health hazards that need further investigation. Studies on well-characterized perylene quinones are urgently needed.

## Genotoxicity

### Mutagenicity in bacterial cells

Several gene mutation assays have been carried out with *Alternaria* toxins (Table 2). The majority of them have been performed by the same research groups. All have been performed in *Salmonella* strains (Ames test), except one that was performed with AME in *E. coli* (An et al. 1989).

AOH induced a weak increase in revertant colonies in TA100 (without and with S9) and TA104 (with S9) and was clearly positive in TA102 in the absence and presence of metabolic activation (Schrader et al. 2001, 2006). AME was also positive, although it elicited only a very weak response in TA102 and TA104 (Schrader et al. 2006); however, it produced a clearly positive result in *E. coli* ND-160 without metabolic activation (An et al. 1989). In this last study, AME was isolated from the fungus by the authors, while in the studies carried out by Schrader (2001, 2006), a commercially available, purified toxin was used. In contrast, Davis and Stack (1994) found negative results for AOH and AME in TA 98, TA 100, TA 1537, and TA 1538 (raw data not shown in the article for 1537 and 1538). The authors hypothesized that the contradictory results in the Ames test were caused by the presence of small amounts of highly mutagenic altertoxins such as ATX-I, ATX-II, and ATX-III, which were consistently positive in all tested strains. Only one study has been carried out for STTX-III (Davis and Stack 1991), showing mutagenicity in TA98 and TA1537 without and with metabolic activation, a weak positive effect in TA100, while there was no effect in TA1535.

On the other hand, TeA, ALT, and TEN were consistently negative in the Ames test (Schrader et al. 2001, 2006). However, it should be noted that the criteria for dose selection were not stated. According to the OECD guideline for the Ames test (OECD 2020), the recommended maximum test concentration for soluble and non-bacteriotoxic substances is 5 mg/plate. In all published Ames studies on *Alternaria* toxins, the test concentrations were far below this recommended concentration (up to 750 µg/plate for AOH and AME, up to 100 µg/plate for TeA, ATX-I, ALT and TEN, up to 60 µg/plate for ATX-II and ATX-III and up to 38.4 µg/plate for STTX-III). Important criteria for the determination of substrate concentrations in the assay are the bacteriotoxicity and solubility in the incubation medium. Toxicity
using the same cell model and comparable dose range. In direct comparison, proliferating cells (HepG2) appear to be more affected by AME than differentiated liver cells (Hep-aRG), raising the question of whether tissue origin or proliferation/differentiation status represents the most important susceptibility factor.

Contradictory results were reported for ALT cytotoxicity in the keratinocyte cell line HaCaT, as well as in HCT116 cells (Table 1). ALS had an overall much higher IC₅₀, between 28.9 and 40 µM. For TeA, IC₅₀ were reported in the range of 70.2–146 µM in most studies. TEN did not show cytotoxic effects in one study in liver cells at levels < 100 µM.

Cytotoxicity data for perylene quinones are still limited. ALP, an analog without epoxide moiety, showed substantial cytotoxic properties in the low micromolar range (IC₅₀ ranging from 2.6 to 6.57 µM). For ATX-I, differing from ALP only by one double bond, the two available reports provided contradictory results. ATX-II represents a perylene quinone with a reactive epoxide moiety. The available studies showed IC₅₀ values ranging from 0.4 to 16.5 µM. The only study using the epoxide-bearing STTX-III showed cytotoxicity above 0.25 µM. Taken together, the cytotoxic properties of ATX-I remain to be clarified, but within the class of perylene quinones cytotoxicity appears not to be limited to the epoxide-bearing analogs since cytotoxic effects have been reported also for ALP. Nevertheless, the limited data available indicate that from the compounds tested so far, ATX-II and STTX-III represent the *Alternaria* toxins with the highest cytotoxic potential. For all studies on *Alternaria* perylene quinones, structural characterization and purity of the compounds are critical factors for the interpretation of the results. So far, these perylene quinones are not accessible via chemical synthesis but need to be isolated from respective fungal cultures. Thereby, the reactivity of the epoxide-bearing analogs (e.g., ATX-II, ATX-III, STTX-III) might lead to unexpected loss of intact test compound, which could underestimate toxicity. On the other hand, traces of these highly toxic epoxide-bearing compounds in preparations of non-epoxide analogs might generate misleading results on apparent toxicity, thus probably overestimating the toxic potential of some isolated analogs.

In summary, most evidence suggests that several *Alternaria* toxins have a cytotoxic potential, some already at nanomolar concentrations. However, some contradictions in the in vitro studies reported indicate that further testing using current guidelines/guidance is needed (e.g., OECD guidelines). Attention should be directed toward the characterization of the toxins present in a cytotoxicity assay, since co-contamination with more than one toxin may introduce bias in the outcome and can cause discrepancies between the studies reported. Most of the test compounds used in the above-cited studies originated from natural sources. Thus,

differences in purity can strongly affect the test results. The combined effects of *Alternaria* toxins that frequently co-occur have been poorly studied, but first results already pointed at the existence of increased health hazards that need further investigation. Studies on well-characterized perylene quinones are urgently needed.

## Genotoxicity

### Mutagenicity in bacterial cells

Several gene mutation assays have been carried out with *Alternaria* toxins (Table 2). The majority of them have been performed by the same research groups. All have been performed in *Salmonella* strains (Ames test), except one that was performed with AME in *E. coli* (An et al. 1989).

AOH induced a weak increase in revertant colonies in TA100 (without and with S9) and TA104 (with S9) and was clearly positive in TA102 in the absence and presence of metabolic activation (Schrader et al. 2001, 2006). AME was also positive, although it elicited only a very weak response in TA102 and TA104 (Schrader et al. 2006); however, it produced a clearly positive result in *E. coli* ND-160 without metabolic activation (An et al. 1989). In this last study, AME was isolated from the fungus by the authors, while in the studies carried out by Schrader (2001, 2006), a commercially available, purified toxin was used. In contrast, Davis and Stack (1994) found negative results for AOH and AME in TA 98, TA 100, TA 1537, and TA 1538 (raw data not shown in the article for 1537 and 1538). The authors hypothesized that the contradictory results in the Ames test were caused by the presence of small amounts of highly mutagenic altertoxins such as ATX-I, ATX-II, and ATX-III, which were consistently positive in all tested strains. Only one study has been carried out for STTX-III (Davis and Stack 1991), showing mutagenicity in TA98 and TA1537 without and with metabolic activation, a weak positive effect in TA100, while there was no effect in TA1535.

On the other hand, TeA, ALT, and TEN were consistently negative in the Ames test (Schrader et al. 2001, 2006). However, it should be noted that the criteria for dose selection were not stated. According to the OECD guideline for the Ames test (OECD 2020), the recommended maximum test concentration for soluble and non-bacteriotoxic substances is 5 mg/plate. In all published Ames studies on *Alternaria* toxins, the test concentrations were far below this recommended concentration (up to 750 µg/plate for AOH and AME, up to 100 µg/plate for TeA, ATX-I, ALT and TEN, up to 60 µg/plate for ATX-II and ATX-III and up to 38.4 µg/plate for STTX-III). Important criteria for the determination of substrate concentrations in the assay are the bacteriotoxicity and solubility in the incubation medium. Toxicity
<table><caption>**Table 2** Summary of published studies reporting bacterial gene mutations assays after exposure to *Alternaria* toxins</caption><thead><tr><th rowspan="2">Bacterial Strain</th><th rowspan="2">Dose (µg/plate)</th><th colspan="2">Effect</th><th rowspan="2">Overall evaluation</th><th rowspan="2">References</th></tr><tr><th>- S9</th><th>+ S9</th></tr></thead><tbody><tr><td colspan="6"><strong>Alternariol (AOH)</strong></td></tr><tr><td>TA98</td><td>50, 125, 250, 500, 750</td><td>-</td><td>-</td><td rowspan="2">Positive (but due to possible contamination with altertoxins)</td><td rowspan="2">Davis and Stack (1994)</td></tr><tr><td>TA100</td><td>50, 125, 250, 500, 750</td><td>-</td><td>-</td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2001)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>Weak +</td><td>Weak +</td></tr><tr><td>TA97</td><td>1, 5, 10, 50<sup>a</sup>, 100<sup>a</sup></td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100<sup>a</sup></td><td>+</td><td>+</td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>-</td><td>weak +</td><td></td><td></td></tr><tr><td colspan="6"><strong>Alternariol monomethyl ether (AME)</strong></td></tr><tr><td>E. coli ND-160</td><td>50, 100</td><td>+</td><td>Not tested</td><td rowspan="2">Weak positive (but due to possible contamination with altertoxins)</td><td>An et al. (1989)</td></tr><tr><td>TA98</td><td>50 to 750 (5 doses)</td><td>-</td><td>-</td><td rowspan="2">Davis and Stack (1994)</td></tr><tr><td>TA100</td><td>50 to 750 (5 doses)</td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2001)</td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td>TA97</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100</td><td>Weak +</td><td>Weak +</td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>-</td><td>Weak +</td><td></td><td></td></tr><tr><td colspan="6"><strong>Tenuazonic acid (TeA)</strong></td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Negative</td><td>Schrader et al. (2001)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA97</td><td>1, 5, 10, 50, 100<sup>a</sup></td><td>-</td><td>-</td><td></td><td></td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100<sup>a</sup></td><td>-</td><td>-</td><td></td><td></td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td></td><td></td></tr><tr><td colspan="6"><strong>Altertoxin (ATX-I)</strong></td></tr><tr><td>TA98</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td><td rowspan="2">Positive</td><td rowspan="2">Stack and Prival (1986)</td></tr><tr><td>TA100</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td></tr><tr><td>TA1537</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td><td></td><td></td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>+</td><td rowspan="2">Schrader et al. (2001)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>Weak +</td><td>Weak +</td></tr><tr><td>TA97</td><td>1, 5, 10, 50, 100</td><td>Weak +</td><td>Weak +</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100</td><td>+</td><td>+</td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>Weak +</td><td>Weak +</td><td></td><td></td></tr><tr><td colspan="6"><strong>Altertoxin II (ATX-II)</strong></td></tr><tr><td>TA98</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td><td rowspan="2">Positive</td><td rowspan="2">Stack and Prival (1986)</td></tr><tr><td>TA100</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td></tr><tr><td>TA 1537</td><td>0.018, 0.06, 0.18, 0.6, 1., 6, 18, 60</td><td>+</td><td>+</td><td></td><td></td></tr><tr><td colspan="6"><strong>Altertoxin III (ATX-III)</strong></td></tr><tr><td>TA98</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td><td rowspan="2">Positive</td><td rowspan="2">Stack and Prival (1986)</td></tr><tr><td>TA100</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td></tr><tr><td>TA 1537</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td><td></td><td></td></tr><tr><td colspan="6"><strong>Altenuene (ALT)</strong></td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Negative</td><td>Schrader et al. (2001)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA97</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td></td><td></td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td></td><td></td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td></td><td></td></tr></tbody></table>
<table><caption>**Table 2** Summary of published studies reporting bacterial gene mutations assays after exposure to *Alternaria* toxins</caption><thead><tr><th rowspan="2">Bacterial Strain</th><th rowspan="2">Dose (µg/plate)</th><th colspan="2">Effect</th><th rowspan="2">Overall evaluation</th><th rowspan="2">References</th></tr><tr><th>- S9</th><th>+ S9</th></tr></thead><tbody><tr><td colspan="6"><strong>Alternariol (AOH)</strong></td></tr><tr><td>TA98</td><td>50, 125, 250, 500, 750</td><td>-</td><td>-</td><td rowspan="2">Positive (but due to possible contamination with altertoxins)</td><td rowspan="2">Davis and Stack (1994)</td></tr><tr><td>TA100</td><td>50, 125, 250, 500, 750</td><td>-</td><td>-</td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2001)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>Weak +</td><td>Weak +</td></tr><tr><td>TA97</td><td>1, 5, 10, 50<sup>a</sup>, 100<sup>a</sup></td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100<sup>a</sup></td><td>+</td><td>+</td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>-</td><td>weak +</td><td></td><td></td></tr><tr><td colspan="6"><strong>Alternariol monomethyl ether (AME)</strong></td></tr><tr><td>E. coli ND-160</td><td>50, 100</td><td>+</td><td>Not tested</td><td rowspan="2">Weak positive (but due to possible contamination with altertoxins)</td><td rowspan="2">An et al. (1989)</td></tr><tr><td>TA98</td><td>50 to 750 (5 doses)</td><td>-</td><td>-</td></tr><tr><td>TA100</td><td>50 to 750 (5 doses)</td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2001)</td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA97</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100</td><td>Weak +</td><td>Weak +</td><td></td><td></td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>-</td><td>Weak +</td><td></td><td></td></tr><tr><td colspan="6"><strong>Tenuazonic acid (TeA)</strong></td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Negative</td><td rowspan="2">Schrader et al. (2001)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td>TA97</td><td>1, 5, 10, 50, 100<sup>a</sup></td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100<sup>a</sup></td><td>-</td><td>-</td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td></td><td></td></tr><tr><td colspan="6"><strong>Altertoxin (ATX-I)</strong></td></tr><tr><td>TA98</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td><td rowspan="2">Positive</td><td rowspan="2">Stack and Prival (1986)</td></tr><tr><td>TA100</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td></tr><tr><td>TA1537</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td><td></td><td></td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>+</td><td rowspan="2">Schrader et al. (2001)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>Weak +</td><td>Weak +</td></tr><tr><td>TA97</td><td>1, 5, 10, 50, 100</td><td>Weak +</td><td>Weak +</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100</td><td>+</td><td>+</td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>Weak +</td><td>Weak +</td><td></td><td></td></tr><tr><td colspan="6"><strong>Altertoxin II (ATX-II)</strong></td></tr><tr><td>TA98</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td><td rowspan="2">Positive</td><td rowspan="2">Stack and Prival (1986)</td></tr><tr><td>TA100</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td></tr><tr><td>TA 1537</td><td>0.018, 0.06, 0.18, 0.6, 1., 6, 18, 60</td><td>+</td><td>+</td><td></td><td></td></tr><tr><td colspan="6"><strong>Altertoxin III (ATX-III)</strong></td></tr><tr><td>TA98</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td><td rowspan="2">Positive</td><td rowspan="2">Stack and Prival (1986)</td></tr><tr><td>TA100</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td></tr><tr><td>TA 1537</td><td>0.018, 0.06, 0.18, 0.6, 1.8, 6, 18, 60</td><td>+</td><td>+</td><td></td><td></td></tr><tr><td colspan="6"><strong>Altenuene (ALT)</strong></td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Negative</td><td rowspan="2">Schrader et al. (2001)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td>TA97</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Schrader et al. (2006)</td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td></td><td></td></tr></tbody></table>
<table><caption>**Table 2** (continued)</caption><thead><tr><th rowspan="2">Bacterial Strain</th><th rowspan="2">Dose (µg/plate)</th><th colspan="2">Effect</th><th rowspan="2">Overall evaluation</th><th rowspan="2">References</th></tr><tr><th>- S9</th><th>+ S9</th></tr></thead><tbody><tr><td colspan="6"><strong>Tentoxin (TEN)</strong></td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Negative</td><td rowspan="2">Schrader et al. (2001)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td>TA97</td><td>1, 5, 10, 50, 100<sup>a</sup></td><td>-</td><td>-</td><td rowspan="3">Positive</td><td rowspan="3">Schrader et al. (2006)</td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td colspan="6"><strong>Stemphyltoxin III (STTX-III)</strong></td></tr><tr><td>TA98</td><td rowspan="2">0.02–38.4 µg/plate (toxicity reported above 38.4 µg)</td><td>+</td><td>+</td><td rowspan="4">Positive</td><td rowspan="4">Davis and Stack (1991)</td></tr><tr><td>TA100</td><td>Weak +</td><td>Weak +</td></tr><tr><td>TA1537</td><td>+</td><td>+</td></tr><tr><td>TA1535</td><td>-</td><td>-</td></tr></tbody></table>

Weak effect refers to a slight increase in revertant colonies but not meeting the twofold criteria for a positive mutagenic response

to the bacterial lawn was reported only in TA97 for AOH (above 50 µg/plate with PBS and 100 µg/plate with S9), TEN and TeA (slight toxicity at 100 µg/plate) and in TA102 for AOH and TeA (both at 100 µg/plate) (Schrader et al. 2006). STTX-III showed toxicity to bacteria at doses higher than 38.4 µg/plate in 4 different strains (Davis and Stack 1991). Precipitation of toxins was not reported in any of the studies. Thus, higher doses might be needed to be tested for toxins showing negative results at non-toxic doses (e.g., ALT, TeA, and TEN).

## Genotoxicity in mammalian cells

A diversity of in vitro genotoxicity assays has been used for investigating the genotoxicity of *Alternaria* toxins AOH, AME, TEA, ATX-II, ATX-III. However, none of the studies reported the use of OECD guidelines (Tables 3 and 4). No studies could be identified addressing the genotoxicity of ATX-I, ATX-III, and TEN.

The studies addressing in vivo the genotoxicity of *Alternaria* toxins are presented in Table 5, frequently being performed according to OECD test guidelines.

Mutagenic effects of *Alternaria* toxins in mammalian cells have been investigated in three published studies, all from the same laboratory. A concentration-dependent increase of the mutant frequency was induced by up to 10 µM chemically synthesized AOH of high purity at the hypoxanthine–guanine phosphoribosyltransferase (HPRT) and the thymidine kinase (TK) gene locus, respectively, in Chinese hamster V79 lung fibroblasts and mouse lymphoma L5178Y tk<sup>+/-</sup> cells (MLC) (Brugger et al. 2006). ATX-II, isolated from *Alternaria alternata*, was shown to be at least a 50-times more potent mutagen in the HPRT assay than AOH and AME (Fleck et al. 2012): AOH caused a

concentration-dependent increase of mutant frequency, starting at 10 µM, while AME increased the frequency of HPRT gene mutations, but not concentration dependent. However, already 0.25 µM ATX-II induced the same mutant frequency as 20 µM AOH or 40 µM AME (Fleck et al. 2012). A similar mutagenic potency was observed for STTX-III that produced an increase in resistant mutants at concentrations above 0.25 µM in the HPRT assay (Fleck et al. 2016). In the same study, the type of DNA damage induced by AOH, ATX-II and STTX-III was investigated, as well as the repair kinetics and their dependence on the status of nucleotide excision repair (NER). AOH-induced damage was removed quickly within 2 h of toxin-free post-incubation, and the repair was independent of the nucleotide excision repair (NER) status of the cells.

As reported, several *Alternaria* toxins cause primary DNA damage in vitro. Most data are available for AOH, AME, and selected altertoxins (Table 4). Single-cell gel electrophoresis (comet assay) is a sensitive, relatively rapid, inexpensive, and technically simple method that is often used to determine xenobiotic-caused DNA damage and is suitable for studies in almost all mammalian cell types. DNA damage (single- and double-strand breaks and other DNA lesions that convert to strand breaks under alkaline conditions) and DNA repair activities can be detected (Møller et al. 2020).

AOH is known to induce DNA strand breaks in various human cell lines in vitro (Fehr et al. 2009; Fleck et al. 2012; Solhaug et al. 2012). Using the comet assay or the alkaline unwinding assay, DNA strand breaks were induced after a short exposure time (1–2 h) at µM concentrations (between 1 and 50 µM) in human HT29 cells (Fehr et al. 2009; Pfeiffer et al. 2007; Schwarz et al. 2012), human A431 cells (Fehr et al. 2009), human HepG2 cells (Pfeiffer et al. 2007), V79

<footnote><sup>a</sup>Reported bacteriotoxic dose in at least one of the conditions tested (PBS or S9)</footnote>
<table><caption>**Table 2** (continued)</caption><thead><tr><th rowspan="2">Bacterial Strain</th><th rowspan="2">Dose (µg/plate)</th><th colspan="2">Effect</th><th rowspan="2">Overall evaluation</th><th rowspan="2">References</th></tr><tr><th>- S9</th><th>+ S9</th></tr></thead><tbody><tr><td colspan="6"><strong>Tentoxin (TEN)</strong></td></tr><tr><td>TA98</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td><td rowspan="2">Negative</td><td rowspan="2">Schrader et al. (2001)</td></tr><tr><td>TA100</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td>TA97</td><td>1, 5, 10, 50, 100<sup>a</sup></td><td>-</td><td>-</td><td rowspan="3">Positive</td><td rowspan="3">Schrader et al. (2006)</td></tr><tr><td>TA102</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td>TA104</td><td>1, 5, 10, 50, 100</td><td>-</td><td>-</td></tr><tr><td colspan="6"><strong>Stemphyltoxin III (STTX-III)</strong></td></tr><tr><td>TA98</td><td>0.02–38.4 µg/plate (toxicity reported above 38.4 µg)</td><td>+</td><td>+</td><td rowspan="4">Positive</td><td rowspan="4">Davis and Stack (1991)</td></tr><tr><td>TA100</td><td></td><td>Weak +</td><td>Weak +</td></tr><tr><td>TA1537</td><td></td><td>+</td><td>+</td></tr><tr><td>TA1535</td><td></td><td>-</td><td>-</td></tr></tbody></table>

Weak effect refers to a slight increase in revertant colonies but not meeting the twofold criteria for a positive mutagenic response

to the bacterial lawn was reported only in TA97 for AOH (above 50 µg/plate with PBS and 100 µg/plate with S9), TEN and TeA (slight toxicity at 100 µg/plate) and in TA102 for AOH and TeA (both at 100 µg/plate) (Schrader et al. 2006). STTX-III showed toxicity to bacteria at doses higher than 38.4 µg/plate in 4 different strains (Davis and Stack 1991). Precipitation of toxins was not reported in any of the studies. Thus, higher doses might be needed to be tested for toxins showing negative results at non-toxic doses (e.g., ALT, TeA, and TEN).

## Genotoxicity in mammalian cells

A diversity of in vitro genotoxicity assays has been used for investigating the genotoxicity of *Alternaria* toxins AOH, AME, TEA, ATX-II, ATX-III. However, none of the studies reported the use of OECD guidelines (Tables 3 and 4). No studies could be identified addressing the genotoxicity of ATX-I, ATX-III, and TEN.

The studies addressing in vivo the genotoxicity of *Alternaria* toxins are presented in Table 5, frequently being performed according to OECD test guidelines.

Mutagenic effects of *Alternaria* toxins in mammalian cells have been investigated in three published studies, all from the same laboratory. A concentration-dependent increase of the mutant frequency was induced by up to 10 µM chemically synthesized AOH of high purity at the hypoxanthine–guanine phosphoribosyltransferase (HPRT) and the thymidine kinase (TK) gene locus, respectively, in Chinese hamster V79 lung fibroblasts and mouse lymphoma L5178Y tk<sup>+/-</sup> cells (MLC) (Brugger et al. 2006). ATX-II, isolated from *Alternaria alternata*, was shown to be at least a 50-times more potent mutagen in the HPRT assay than AOH and AME (Fleck et al. 2012): AOH caused a

concentration-dependent increase of mutant frequency, starting at 10 µM, while AME increased the frequency of HPRT gene mutations, but not concentration dependent. However, already 0.25 µM ATX-II induced the same mutant frequency as 20 µM AOH or 40 µM AME (Fleck et al. 2012). A similar mutagenic potency was observed for STTX-III that produced an increase in resistant mutants at concentrations above 0.25 µM in the HPRT assay (Fleck et al. 2016). In the same study, the type of DNA damage induced by AOH, ATX-II and STTX-III was investigated, as well as the repair kinetics and their dependence on the status of nucleotide excision repair (NER). AOH-induced damage was removed quickly within 2 h of toxin-free post-incubation, and the repair was independent of the nucleotide excision repair (NER) status of the cells.

As reported, several *Alternaria* toxins cause primary DNA damage in vitro. Most data are available for AOH, AME, and selected altertoxins (Table 4). Single-cell gel electrophoresis (comet assay) is a sensitive, relatively rapid, inexpensive, and technically simple method that is often used to determine xenobiotic-caused DNA damage and is suitable for studies in almost all mammalian cell types. DNA damage (single- and double-strand breaks and other DNA lesions that convert to strand breaks under alkaline conditions) and DNA repair activities can be detected (Møller et al. 2020).

AOH is known to induce DNA strand breaks in various human cell lines in vitro (Fehr et al. 2009; Fleck et al. 2012; Solhaug et al. 2012). Using the comet assay or the alkaline unwinding assay, DNA strand breaks were induced after a short exposure time (1–2 h) at µM concentrations (between 1 and 50 µM) in human HT29 cells (Fehr et al. 2009; Pfeiffer et al. 2007; Schwarz et al. 2012), human A431 cells (Fehr et al. 2009), human HepG2 cells (Pfeiffer et al. 2007), V79

<footnote><sup>a</sup>Reported bacteriotoxic dose in at least one of the conditions tested (PBS or S9)</footnote>
<table><caption>**Table 3** Summary of published in vitro genotoxicity studies in mammalian cells</caption><thead><tr><th>Cell line</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>S9 (Y/N)</th><th>Effect</th><th>References</th></tr></thead><tbody><tr><td colspan="6"><strong>Alternariol (AOH)</strong></td></tr><tr><td colspan="6">HPRT and XPRT mutation assay</td></tr><tr><td>V79</td><td>5, 10, 15, 20, 25, 30</td><td>24</td><td>N</td><td>Positive ≥ 10 µM (HPRT assay)</td><td>Brugger et al. (2006)</td></tr><tr><td>V79</td><td>5, 10, 15, 20</td><td>24</td><td>N</td><td>Positive ≥ 10 µM (HPRT assay)</td><td>Fleck et al. (2012)</td></tr><tr><td colspan="6">TK gene mutation assay</td></tr><tr><td>L5178Y tk+/-</td><td>5, 10, 15, 20, 25, 30</td><td>24</td><td>N</td><td>Positive ≥ 10 µM</td><td>Brugger et al. (2006)</td></tr><tr><td colspan="6">Micronucleus assay</td></tr><tr><td>RAW264.7</td><td>30</td><td>48</td><td>N</td><td>Positive</td><td>Solhaug et al. (2013)</td></tr><tr><td>Ishikawa</td><td>1; 2.5; 5; 10</td><td>48</td><td>N</td><td>Positive ≥ 5 µM</td><td>Lehmann et al. (2006)</td></tr><tr><td>V79</td><td>2.5; 5; 10; 25; 50</td><td>6</td><td>N</td><td>Positive ≥ 5 µM</td><td>Lehmann et al. (2006)</td></tr><tr><td colspan="6"><strong>Alternariol monomethyl ether (AME)</strong></td></tr><tr><td colspan="6">HPRT and XPRT mutation assay</td></tr><tr><td>V79</td><td>10, 20, 30, 40</td><td>24</td><td>N</td><td>Positive ≥ 20 µM (HPRT assay)</td><td>Fleck et al. (2012)</td></tr><tr><td colspan="6"><strong>Tenuazonic acid (TeA)</strong></td></tr><tr><td colspan="6">HPRT and XPRT mutation assay</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.50, 0.75</td><td>24</td><td>N</td><td>Positive ≥ 0.25 µM (HPRT assay)</td><td>Fleck et al. (2012)</td></tr><tr><td colspan="6"><strong>Altertoxin II (ATX-II)</strong></td></tr><tr><td colspan="6">HPRT and XPRT mutation assay</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.50, 0.75</td><td>24</td><td>N</td><td>Positive ≥ 0.25 µM (HPRT assay)</td><td>Fleck et al. (2012)</td></tr><tr><td colspan="6"><strong>Stemphyltoxin (STTX-III)</strong></td></tr><tr><td colspan="6">HPRT and XPRT mutation assay</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.5, 0.75</td><td>24</td><td>N</td><td>Positive ≥ 0.25 µM (HPRT assay)</td><td>Fleck et al. (2016)</td></tr></tbody></table>

V79 lung Chinese hamster fibroblasts, L5178Y tk+/- mouse lymphoma cells, RAW 264.7 Murine macrophage cells, Ishikawa endometrial adenocarcinoma cells, HPRT hypoxanthine-guanine phosphoribosyltransferase, TK thymidine kinase

fibroblasts (Fleck et al. 2012; Pfeiffer et al. 2007) and in the murine macrophages RAW 264.7 (Solhaug et al. 2012). In contrast, DNA damage was not observed after prolonged incubation (3 h, 24 h) with up to 50 µM AOH in HT29 cells (Pfeiffer et al. 2007; Tiessen et al. 2013b). The authors suggested that AOH toxicity was decreased by rapid biotransformation reactions such as glucuronidation (Pfeiffer et al. 2009b) and glutathione (GSH) conjugation (Tiessen et al. 2013b) which protected the cells from DNA damage. On the contrary, AOH has been shown to induce DNA damage after 24 h of exposure in HepG2 cells (Pfeiffer et al. 2007) as well as in RAW 264.7, Caco-2 and Hek239 (Fernández-Blanco et al. 2015; Solhaug et al. 2012; Tran et al. 2020). Nevertheless, due to the prolonged incubation period, it cannot be excluded that the observed DNA damage does not arise from direct genotoxic mechanisms but reflects DNA degrading effects due to cell death. Furthermore, 30 µM AOH induced oxidative DNA damage in RAW 264.7 cells after 2-h exposure as assessed by a modified comet assay (Solhaug et al. 2012). In this version of the comet assay, among other purine base modifications, 8-oxoGua, a common product of oxidative DNA damage in cells, is converted to single-strand breaks by the addition of formamidopyrimidine-DNA-glycosylase (fpg) enzyme (Collins et al. 2008).

The study of Solhaug et al. suggests that AOH enhanced the amount of reactive oxidative species (ROS) and oxidation of DNA bases (Solhaug et al. 2012). Interestingly, oxidative DNA damage was not detected in the same cell line after prolonged exposure, i.e., after 24 h with up to 30 µM, whereas DNA strand breaks were generated in the classical comet assay (Solhaug et al. 2012). Furthermore, up to 50 µM AOH did not induce fpg-sensitive sites in HT29 and A431 cells after 1-h exposure, suggesting that oxidative stress may not play a predominant role in the induction of DNA damage (Tiessen et al. 2013a; Solhaug et al. 2012). Although most in vitro genotoxicity tests were positive, AOH genotoxicity has rarely been studied in vivo. A study in orally treated NMRI mice showed that AOH did not cause DNA damage in the liver at 2000 mg/kg bodyweight (bw) regardless of sex, neither after a single dose nor with three repeated doses (Schuchardt et al. 2014). Recently, it was demonstrated that up to 22 µg AOH/kg b.w. orally for 28 days did not cause DNA damage in peripheral blood and liver cells of male Sprague Dawley rats as determined by the comet assay, probably because the systemic bioavailability of AOH is very low (Miao et al. 2022). In contrast, a single oral dose of 10 mg AOH/kg b.w. in Wistar rats significantly increased DNA damage in acinar cells (Samak et al. 2019).
<table><caption>**Table 3** Summary of published in vitro genotoxicity studies in mammalian cells</caption><thead><tr><th>Cell line</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>S9 (Y/N)</th><th>Effect</th><th>References</th></tr></thead><tbody><tr><td colspan="6"><strong>Alternariol (AOH)</strong></td></tr><tr><td colspan="6">HPRT and XPRT mutation assay</td></tr><tr><td>V79</td><td>5, 10, 15, 20, 25, 30</td><td>24</td><td>N</td><td>Positive ≥ 10 µM (HPRT assay)</td><td>Brugger et al. (2006)</td></tr><tr><td>V79</td><td>5, 10, 15, 20</td><td>24</td><td>N</td><td>Positive ≥ 10 µM (HPRT assay)</td><td>Fleck et al. (2012)</td></tr><tr><td colspan="6">TK gene mutation assay</td></tr><tr><td>L5178Y tk+/-</td><td>5, 10, 15, 20, 25, 30</td><td>24</td><td>N</td><td>Positive ≥ 10 µM</td><td>Brugger et al. (2006)</td></tr><tr><td colspan="6">Micronucleus assay</td></tr><tr><td>RAW264.7</td><td>30</td><td>48</td><td>N</td><td>Positive</td><td>Solhaug et al. (2013)</td></tr><tr><td>Ishikawa</td><td>1; 2.5; 5; 10</td><td>48</td><td>N</td><td>Positive ≥ 5 µM</td><td>Lehmann et al. (2006)</td></tr><tr><td>V79</td><td>2.5; 5; 10; 25; 50</td><td>6</td><td>N</td><td>Positive ≥ 5 µM</td><td>Lehmann et al. (2006)</td></tr><tr><td colspan="6"><strong>Alternariol monomethyl ether (AME)</strong></td></tr><tr><td colspan="6">HPRT and XPRT mutation assay</td></tr><tr><td>V79</td><td>10, 20, 30, 40</td><td>24</td><td>N</td><td>Positive ≥ 20 µM (HPRT assay)</td><td>Fleck et al. (2012)</td></tr><tr><td colspan="6"><strong>Tenuazonic acid (TeA)</strong></td></tr><tr><td colspan="6">HPRT and XPRT mutation assay</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.50, 0.75</td><td>24</td><td>N</td><td>Positive ≥ 0.25 µM (HPRT assay)</td><td>Fleck et al. (2012)</td></tr><tr><td colspan="6"><strong>Altertoxin II (ATX-II)</strong></td></tr><tr><td colspan="6">HPRT and XPRT mutation assay</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.50, 0.75</td><td>24</td><td>N</td><td>Positive ≥ 0.25 µM (HPRT assay)</td><td>Fleck et al. (2012)</td></tr><tr><td colspan="6"><strong>Stemphyltoxin (STTX-III)</strong></td></tr><tr><td colspan="6">HPRT and XPRT mutation assay</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.5, 0.75</td><td>24</td><td>N</td><td>Positive ≥ 0.25 µM (HPRT assay)</td><td>Fleck et al. (2016)</td></tr></tbody></table>

V79 lung Chinese hamster fibroblasts, L5178Y tk+/- mouse lymphoma cells, RAW 264.7 Murine macrophage cells, Ishikawa endometrial adenocarcinoma cells, HPRT hypoxanthine-guanine phosphoribosyltransferase, TK thymidine kinase

fibroblasts (Fleck et al. 2012; Pfeiffer et al. 2007) and in the murine macrophages RAW 264.7 (Solhaug et al. 2012). In contrast, DNA damage was not observed after prolonged incubation (3 h, 24 h) with up to 50 µM AOH in HT29 cells (Pfeiffer et al. 2007; Tiessen et al. 2013b). The authors suggested that AOH toxicity was decreased by rapid biotransformation reactions such as glucuronidation (Pfeiffer et al. 2009b) and glutathione (GSH) conjugation (Tiessen et al. 2013b) which protected the cells from DNA damage. On the contrary, AOH has been shown to induce DNA damage after 24 h of exposure in HepG2 cells (Pfeiffer et al. 2007) as well as in RAW 264.7, Caco-2 and Hek239 (Fernández-Blanco et al. 2015; Solhaug et al. 2012; Tran et al. 2020). Nevertheless, due to the prolonged incubation period, it cannot be excluded that the observed DNA damage does not arise from direct genotoxic mechanisms but reflects DNA degrading effects due to cell death. Furthermore, 30 µM AOH induced oxidative DNA damage in RAW 264.7 cells after 2-h exposure as assessed by a modified comet assay (Solhaug et al. 2012). In this version of the comet assay, among other purine base modifications, 8-oxoGua, a common product of oxidative DNA damage in cells, is converted to single-strand breaks by the addition of formamidopyrimidine-DNA-glycosylase (fpg) enzyme (Collins et al. 2008).

The study of Solhaug et al. suggests that AOH enhanced the amount of reactive oxidative species (ROS) and oxidation of DNA bases (Solhaug et al. 2012). Interestingly, oxidative DNA damage was not detected in the same cell line after prolonged exposure, i.e., after 24 h with up to 30 µM, whereas DNA strand breaks were generated in the classical comet assay (Solhaug et al. 2012). Furthermore, up to 50 µM AOH did not induce fpg-sensitive sites in HT29 and A431 cells after 1-h exposure, suggesting that oxidative stress may not play a predominant role in the induction of DNA damage (Tiessen et al. 2013a; Solhaug et al. 2012). Although most in vitro genotoxicity tests were positive, AOH genotoxicity has rarely been studied in vivo. A study in orally treated NMRI mice showed that AOH did not cause DNA damage in the liver at 2000 mg/kg bodyweight (bw) regardless of sex, neither after a single dose nor with three repeated doses (Schuchardt et al. 2014). Recently, it was demonstrated that up to 22 µg AOH/kg b.w. orally for 28 days did not cause DNA damage in peripheral blood and liver cells of male Sprague Dawley rats as determined by the comet assay, probably because the systemic bioavailability of AOH is very low (Miao et al. 2022). In contrast, a single oral dose of 10 mg AOH/kg b.w. in Wistar rats significantly increased DNA damage in acinar cells (Samak et al. 2019).
<table><caption>**Table 4** Summary of published in vitro studies with non-OECD genotoxicity assays in mammalian cells</caption><thead><tr><th>Cell line</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>S9 (Y/N)</th><th>Effect</th><th>References</th></tr></thead><tbody><tr><td colspan="6"><strong>Alternariol (AOH)</strong></td></tr><tr><td colspan="6"><strong>γH2AX assay</strong></td></tr><tr><td>PNT1A c</td><td>0.1, 10</td><td>no info</td><td>N</td><td>Positive (10 µM)<br>(Mouse®Multi-Color DNA<br>Damage Kit)</td><td>Kowalska et al. (2021b)</td></tr><tr><td>HepG2</td><td>0.1, 10, 100</td><td>4</td><td>Y</td><td>- S9: positive (100 µM)<br>+ S9: complete</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>Human primary mac-<br>rophages</td><td>15, 30, 60<br>15, 30, 60</td><td>6<br>24</td><td>N<br>N</td><td>Positive<br>Positive (&gt; 30 µM)</td><td>Solhaug et al. (2015)</td></tr><tr><td>RAW 264.7</td><td>30</td><td>6, 24, 48</td><td>N</td><td>Positive</td><td>Solhaug et al. (2012)</td></tr><tr><td colspan="6"><strong>Comet assay</strong></td></tr><tr><td>A431</td><td>0.1, 1, 10, 25, 50</td><td>1</td><td>N</td><td>Standard comet assay; posi-<br>tive; ≥ 1 µM<br>Fpg comet assay; nega-<br>tive; ≥ 1 µM (no significant<br>difference without and<br>with fpg)</td><td>Fehr et al. (2009)</td></tr><tr><td>A431</td><td>0.1, 1, 10, 50</td><td>1</td><td>N</td><td>Neutral comet<br>assay; ≥ 10 µM</td><td>Fehr et al. (2010)</td></tr><tr><td>Caco-2</td><td>15, 30, 60</td><td>24</td><td>N</td><td>Standard comet assay; posi-<br>tive ≥ 15 µM</td><td>Fernández-Blanco et al.<br>(2015)</td></tr><tr><td>HEK 293 T</td><td>25</td><td>24</td><td>N</td><td>Standard comet assay;<br>positive</td><td>Tran et al. (2020)</td></tr><tr><td>HEK293, transfected;<br>GFP, GFP-TDP1<sup>H263A</sup>,<br>GFP-TDP1</td><td>0.1, 1, 10, and 50</td><td>1</td><td>N</td><td>Standard comet assay;<br>positive; ≥ 1 µM; GFP,<br>GFP-TDP1, ≥ 10 µM GFP-<br>TDP1<sup>H263A</sup></td><td>Fehr et al. (2010)</td></tr><tr><td>HT29</td><td>0.1, 1, 10, 50</td><td>1</td><td>N</td><td>Standard comet assay; posi-<br>tive ≥ 10 µM<br>Fpg comet assay; negative</td><td>Schwarz et al. (2012)</td></tr><tr><td>HT29</td><td>0.1, 0.5, 1, 10, 25, 50</td><td>1</td><td>N</td><td>Standard comet assay; posi-<br>tive ≥ 0.5 µM<br>Fpg comet assay; negative</td><td>Tiessen et al. (2013b)</td></tr><tr><td>HT29</td><td>0.1, 1, 10, 25, 50, and 100</td><td>1</td><td>N</td><td>Standard comet assay; posi-<br>tive; ≥ 1 µM<br>Fpg comet assay; Negative</td><td>Fehr et al. (2009)</td></tr><tr><td>HT29</td><td>50</td><td>1</td><td>N</td><td>Standard comet assay;<br>positive<br>Fpg comet assay; positive</td><td>Aichinger et al. (2017)</td></tr><tr><td>HT29</td><td>50</td><td>3</td><td>N</td><td>Standard comet assay;<br>negative<br>Fpg comet assay; negative</td><td>Tiessen et al. (2013a)</td></tr><tr><td>KYSE510</td><td>1, 10, 50</td><td>1</td><td>N</td><td>Standard comet assay; only<br>at 50 µM positive<br>Fpg comet assay; negative</td><td>Tiessen et al. (2017)</td></tr><tr><td>RAW 264.7</td><td>30</td><td>2</td><td>N</td><td>Standard comet assay;<br>positive<br>Fpg comet assay; positive</td><td>Solhaug et al. (2012)</td></tr><tr><td>RAW 264.7</td><td>15 and 30</td><td>24</td><td>N</td><td>Standard comet assay; posi-<br>tive at 30 µM<br>Fpg comet assay; negative</td><td>Solhaug et al. (2012)</td></tr><tr><td colspan="6"><strong>Alkaline unwinding assay</strong></td></tr><tr><td>Caco-2</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>HepG2</td><td>12.5, 25, 50</td><td>1</td><td>N</td><td>≥ 12.5 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>HepG2</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>HT29</td><td>1, 6.25, 12.5, 25,</td><td>1</td><td>N</td><td>≥ 6.25 µM</td><td>Pfeiffer et al. (2007)</td></tr></tbody></table>
<table><caption>**Table 4** Summary of published in vitro studies with non-OECD genotoxicity assays in mammalian cells</caption><thead><tr><th>Cell line</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>S9 (Y/N)</th><th>Effect</th><th>References</th></tr></thead><tbody><tr><td colspan="6"><strong>Alternariol (AOH)</strong></td></tr><tr><td colspan="6"><strong>γH2AX assay</strong></td></tr><tr><td>PNT1A c</td><td>0.1, 10</td><td>no info</td><td>N</td><td>Positive (10 µM) (Mouse®Multi-Color DNA Damage Kit)</td><td>Kowalska et al. (2021b)</td></tr><tr><td>HepG2</td><td>0.1, 10, 100</td><td>4</td><td>Y</td><td>- S9: positive (100 µM) +S9: complete</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td>Human primary macrophages</td><td>15, 30, 60</td><td>6</td><td>N</td><td>Positive</td><td>Solhaug et al. (2015)</td></tr><tr><td>RAW 264.7</td><td>15, 30, 60</td><td>24</td><td>N</td><td>Positive (&gt; 30 µM)</td><td>Solhaug et al. (2012)</td></tr><tr><td colspan="6"><strong>Comet assay</strong></td></tr><tr><td>A431</td><td>0.1, 1, 10, 25, 50</td><td>1</td><td>N</td><td>Standard comet assay; positive; ≥ 1 µM<br>Fpg comet assay; negative; ≥ 1 µM (no significant difference without and with fpg)</td><td>Fehr et al. (2009)</td></tr><tr><td>A431</td><td>0.1, 1, 10, 50</td><td>1</td><td>N</td><td>Neutral comet assay; ≥ 10 µM</td><td>Fehr et al. (2010)</td></tr><tr><td>Caco-2</td><td>15, 30, 60</td><td>24</td><td>N</td><td>Standard comet assay; positive ≥ 15 µM</td><td>Fernández-Blanco et al. (2015)</td></tr><tr><td>HEK 293 T</td><td>25</td><td>24</td><td>N</td><td>Standard comet assay; positive</td><td>Tran et al. (2020)</td></tr><tr><td>HEK293, transfected; GFP, GFP-TDP1<sup>H263A</sup>, GFP-TDP1</td><td>0.1, 1, 10, and 50</td><td>1</td><td>N</td><td>Standard comet assay; positive; ≥ 1 µM; GFP, GFP-TDP1, ≥ 10 µM GFP-TDP1<sup>H263A</sup></td><td>Fehr et al. (2010)</td></tr><tr><td>HT29</td><td>0.1, 1, 10, 50</td><td>1</td><td>N</td><td>Standard comet assay; positive ≥ 10 µM<br>Fpg comet assay; negative</td><td>Schwarz et al. (2012)</td></tr><tr><td>HT29</td><td>0.1, 0.5, 1, 10, 25, 50</td><td>1</td><td>N</td><td>Standard comet assay; positive ≥ 0.5 µM<br>Fpg comet assay; negative</td><td>Tiessen et al. (2013b)</td></tr><tr><td>HT29</td><td>0.1, 1, 10, 25, 50, and 100</td><td>1</td><td>N</td><td>Standard comet assay; positive; ≥ 1 µM<br>Fpg comet assay; negative</td><td>Fehr et al. (2009)</td></tr><tr><td>HT29</td><td>50</td><td>1</td><td>N</td><td>Standard comet assay; positive<br>Fpg comet assay; positive</td><td>Aichinger et al. (2017)</td></tr><tr><td>HT29</td><td>50</td><td>3</td><td>N</td><td>Standard comet assay; negative<br>Fpg comet assay; negative</td><td>Tiessen et al. (2013a)</td></tr><tr><td>KYSE510</td><td>1, 10, 50</td><td>1</td><td>N</td><td>Standard comet assay; only at 50 µM positive<br>Fpg comet assay; negative</td><td>Tiessen et al. (2017)</td></tr><tr><td>RAW 264.7</td><td>30</td><td>2</td><td>N</td><td>Standard comet assay; positive<br>Fpg comet assay; positive</td><td>Solhaug et al. (2012)</td></tr><tr><td>RAW 264.7</td><td>15 and 30</td><td>24</td><td>N</td><td>Standard comet assay; positive at 30 µM<br>Fpg comet assay; negative</td><td>Solhaug et al. (2012)</td></tr><tr><td colspan="6"><strong>Alkaline unwinding assay</strong></td></tr><tr><td>Caco-2</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>HepG2</td><td>12.5, 25, 50</td><td>1</td><td>N</td><td>≥ 12.5 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>HepG2</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>HT29</td><td>1, 6.25, 12.5, 25,</td><td>1</td><td>N</td><td>≥ 6.25 µM</td><td>Pfeiffer et al. (2007)</td></tr></tbody></table>
<table><caption>**Table 4 (continued)**</caption><thead><tr><th>Cell line</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>S9 (Y/N)</th><th>Effect</th><th>References</th></tr></thead><tbody><tr><td>HT29</td><td>5, 10, 25</td><td>24</td><td>N</td><td>Negative</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>V79</td><td>12.5, 25, 50</td><td>1</td><td>N</td><td>≥ 12.5 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.5, 1, 5, 10, 20</td><td>1.5</td><td>N</td><td>≥ 5 µM</td><td>Fleck et al. (2012)</td></tr><tr><td>V79</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td colspan="6"><strong>Alternariol monomethyl ether (AME)</strong></td></tr><tr><td colspan="6"><strong>H2AX assay</strong></td></tr><tr><td>HepG2</td><td>0.1, 10, 100</td><td>4</td><td>Y</td><td>Higher levels of γH2AX only detected at high-est concentration in the absence of S9</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td colspan="6"><strong>Comet assay</strong></td></tr><tr><td>A431</td><td>0.1, 1, 10, 50</td><td>1</td><td>N</td><td>Standard comet assay; positive; ≥ 1 µM<br>Fpg comet assay; negative</td><td>Fehr et al. (2009)</td></tr><tr><td>A431</td><td>0.1, 1, 10 and 50</td><td>1</td><td>N</td><td>Neutral comet assay; ≥ 10 µM positive</td><td>Fehr et al. (2010)</td></tr><tr><td>HEK 293 T</td><td>25</td><td>24</td><td>N</td><td>Standard comet assay; positive</td><td>Tran et al. (2020)</td></tr><tr><td>HT29</td><td>1, 10, 25 and 50</td><td>1</td><td>N</td><td>Standard comet assay; positive; ≥ 10 µM<br>Fpg comet assay; negative</td><td>Fehr et al. (2009)</td></tr><tr><td>HT29</td><td>0.1, 1, 10, 50</td><td>3</td><td>N</td><td>Standard comet assay; negative<br>Fpg comet assay; negative</td><td>Tiessen et al. (2013a)</td></tr><tr><td>KYSE510</td><td>10, 50</td><td>1</td><td>N</td><td>Standard comet assay; negative<br>Fpg comet assay; negative</td><td>Tiessen et al. (2017)</td></tr><tr><td colspan="6"><strong>Alkaline unwinding assay</strong></td></tr><tr><td>HepG2</td><td>6.25, 12.5, 25, 50</td><td>1</td><td>N</td><td>≥ 6.25 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>HepG2</td><td>5, 10, 25</td><td>24</td><td>N</td><td>≥ 5 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>HT29</td><td>1, 6.25, 12.5, 25</td><td>1</td><td>N</td><td>≥ 6.25 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>HT29</td><td>5, 10, 25</td><td>24</td><td>N</td><td>Negative</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>V79</td><td>6.25, 12.5, 25, 50</td><td>1</td><td>N</td><td>≥ 6.25 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.5, 1, 5, 10, 20</td><td>1,5</td><td>N</td><td>≥ 0.5 µM</td><td>Fleck et al. (2012)</td></tr><tr><td colspan="6"><strong>Tenuazonic acid (TeA)</strong></td></tr><tr><td colspan="6"><strong>H2AX assay</strong></td></tr><tr><td>Hep G2</td><td>0.1, 10, 100 µM</td><td>4</td><td>Y</td><td>Negative with or without S9</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td colspan="6"><strong>Comet assay</strong></td></tr><tr><td>HT29</td><td>0.2, 2, 20, 200</td><td>1</td><td>N</td><td>Standard comet assay; negative<br>Fpg comet assay; negative</td><td>Schwarz et al. (2012)</td></tr><tr><td colspan="6"><strong>Altertoxin I (ATX-I)</strong></td></tr><tr><td colspan="6"><strong>Alkaline unwinding assay</strong></td></tr><tr><td>Caco-2</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>HepG2</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>V79</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td colspan="6"><strong>Altertoxin II (ATX-II)</strong></td></tr><tr><td colspan="6"><strong>Comet assay</strong></td></tr><tr><td>HT29</td><td>0.01, 0.05, 0.1, 0.02, 0.05, 1</td><td>1</td><td>N</td><td>Standard comet assay; positive ≥ 0.1 µM<br>Fpg comet assay; positive ≥ 0.05 µM</td><td>Tiessen et al. (2013b)</td></tr></tbody></table>

<figure><img src="image_7.png" /></figure>
<table><caption>**Table 4 (continued)**</caption><thead><tr><th>Cell line</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>S9 (Y/N)</th><th>Effect</th><th>References</th></tr></thead><tbody><tr><td>HT29</td><td>5, 10, 25</td><td>24</td><td>N</td><td>Negative</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>V79</td><td>12.5, 25, 50</td><td>1</td><td>N</td><td>≥ 12.5 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.5, 1, 5, 10, 20</td><td>1.5</td><td>N</td><td>≥ 5 µM</td><td>Fleck et al. (2012)</td></tr><tr><td>V79</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td colspan="6">Alternariol monomethyl ether (AME)</td></tr><tr><td colspan="6">H2AX assay</td></tr><tr><td>HepG2</td><td>0.1, 10, 100</td><td>4</td><td>Y</td><td>Higher levels of γH2AX only detected at high-est concentration in the absence of S9</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td colspan="6">Comet assay</td></tr><tr><td>A431</td><td>0.1, 1, 10, 50</td><td>1</td><td>N</td><td>Standard comet assay; positive; ≥ 1 µM<br>Fpg comet assay; negative</td><td>Fehr et al. (2009)</td></tr><tr><td>A431</td><td>0.1, 1, 10 and 50</td><td>1</td><td>N</td><td>Neutral comet assay; ≥ 10 µM positive</td><td>Fehr et al. (2010)</td></tr><tr><td>HEK 293 T</td><td>25</td><td>24</td><td>N</td><td>Standard comet assay; positive</td><td>Tran et al. (2020)</td></tr><tr><td>HT29</td><td>1, 10, 25 and 50</td><td>1</td><td>N</td><td>Standard comet assay; positive; ≥ 10 µM<br>Fpg comet assay; negative</td><td>Fehr et al. (2009)</td></tr><tr><td>HT29</td><td>0.1, 1, 10, 50</td><td>3</td><td>N</td><td>Standard comet assay; negative<br>Fpg comet assay; negative</td><td>Tiessen et al. (2013a)</td></tr><tr><td>KYSE510</td><td>10, 50</td><td>1</td><td>N</td><td>Standard comet assay; negative<br>Fpg comet assay; negative</td><td>Tiessen et al. (2017)</td></tr><tr><td colspan="6">Alkaline unwinding assay</td></tr><tr><td>HepG2</td><td>6.25, 12.5, 25, 50</td><td>1</td><td>N</td><td>≥ 6.25 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>HepG2</td><td>5, 10, 25</td><td>24</td><td>N</td><td>≥ 5 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>HT29</td><td>1, 6.25, 12.5, 25</td><td>1</td><td>N</td><td>≥ 6.25 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>HT29</td><td>5, 10, 25</td><td>24</td><td>N</td><td>Negative</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>V79</td><td>6.25, 12.5, 25, 50</td><td>1</td><td>N</td><td>≥ 6.25 µM</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.5, 1, 5, 10, 20</td><td>1.5</td><td>N</td><td>≥ 0.5 µM</td><td>Fleck et al. (2012)</td></tr><tr><td colspan="6">Tenuazonic acid (TeA)</td></tr><tr><td colspan="6">H2AX assay</td></tr><tr><td>Hep G2</td><td>0.1, 10, 100 µM</td><td>4</td><td>Y</td><td>Negative with or without S9</td><td>Hessel-Pras et al. (2019)</td></tr><tr><td colspan="6">Comet assay</td></tr><tr><td>HT29</td><td>0.2, 2, 20, 200</td><td>1</td><td>N</td><td>Standard comet assay; negative<br>Fpg comet assay; negative</td><td>Schwarz et al. (2012)</td></tr><tr><td colspan="6">Altertoxin I (ATX-I)</td></tr><tr><td colspan="6">Alkaline unwinding assay</td></tr><tr><td>Caco-2</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>HepG2</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>V79</td><td>10</td><td>1.5</td><td>N</td><td>Positive</td><td>Fleck et al. (2014a, b)</td></tr><tr><td colspan="6">Altertoxin II (ATX-II)</td></tr><tr><td colspan="6">Comet assay</td></tr><tr><td>HT29</td><td>0.01, 0.05, 0.1, 0.02, 0.05, 1</td><td>1</td><td>N</td><td>Standard comet assay; positive ≥ 0.1 µM<br>Fpg comet assay; positive ≥ 0.05 µM</td><td>Tiessen et al. (2013b)</td></tr></tbody></table>

<figure><img src="image_7.png" /></figure>
<table><caption>**Table 4** (continued)</caption><thead><tr><th>Cell line</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>S9 (Y/N)</th><th>Effect</th><th>References</th></tr></thead><tbody><tr><td>HT29</td><td>1</td><td>1</td><td>N</td><td>Standard comet assay; positive<br>Fpg comet assay; positive</td><td>Aichinger et al. (2018)</td></tr><tr><td>HT29</td><td>0.01, 0.05, 0.1, 0.2, 0.5, 1</td><td>1</td><td>N</td><td>Standard comet assay; positive ≥ 0.1 µM<br>Fpg comet assay; positive ≥ 0.05 µM</td><td>Schwarz et al. (2012)</td></tr><tr><td>HT29</td><td>0.01, 0.05, 0.1, 0.2, 0.5, 1</td><td>24</td><td>N</td><td>Standard comet assay; positive ≥ 0.1 µM<br>Fpg comet assay; positive ≥ 0.05 µM</td><td>Schwarz et al. (2012)</td></tr><tr><td>Ishikawa cells</td><td>1</td><td>1</td><td>N</td><td>Standard comet assay; positive<br>Fpg comet assay; positive</td><td>Aichinger et al. (2022a)</td></tr><tr><td colspan="6"><strong>Alkaline unwinding assay</strong></td></tr><tr><td>Caco-2</td><td>0.25, 0.5, 1</td><td>1.5</td><td>N</td><td>≥ 0.25 µM</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>HepG2</td><td>0.25, 0.5, 1</td><td>1.5</td><td>N</td><td>≥ 0.25 µM</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.5, 1, 5, 10, 20</td><td>1.5</td><td>N</td><td>≥ 0.1 µM</td><td>Fleck et al. (2012)</td></tr><tr><td>V79</td><td>0.25, 0.5, 1</td><td>1.5</td><td>N</td><td>≥ 0.25 µM</td><td>Fleck et al. (2014a, b)</td></tr><tr><td colspan="6"><strong>Tentoxin (TEN)</strong></td></tr><tr><td colspan="6"><strong>Comet assay</strong></td></tr><tr><td>HEK 293 T</td><td>25</td><td>24</td><td>N</td><td>Standard comet assay; negative</td><td>Tran et al. (2020)</td></tr></tbody></table>

*Cell lines* PNT1A- human primary prostate epithelial cells, *HepG2* human liver carcinoma, *A431* human vulva carcinoma, *Caco-2* human colon carcinoma, *HEK 293T* human embryonal kidney, *HT29* human colorectal adenocarcinoma, *KYSE510* human esophageal squamous carcinoma, *RAW264.7* mouse macrophage, *V79* lung Chinese hamster fibroblasts, *Ishikawa* endometrial adenocarcinoma cells

AME induced DNA strand breaks in HT29 and A431 cells at concentrations ≥ 10 and ≥ 1 µM, respectively, after 1 h of exposure as assessed in the alkaline comet assay (Fehr et al. 2009), indicating a substantial genotoxic potential. The rate of DNA strand breaks induced by AME did not statistically significantly differ from fpg-treated cells, suggesting that AME does not cause oxidative DNA damage (Fehr et al. 2009). In the alkaline unwinding assay, AME showed slightly but not significantly lower DNA strand-breaking activities as compared to AOH in both cell lines (Pfeiffer et al. 2007). Furthermore, using the same assay, a dose-dependent increase (> 6.25 µM) of DNA strand breaks in HT29, HepG2, and V79 cells after 1 h was revealed (Pfeiffer et al. 2007). At concentrations above 10 µM, AME induced DNA double-strand breaks in A431 cells in the comet assay under neutral pH conditions after 1 h (Fehr et al. 2010). Similarly, using the alkaline unwinding assay, it was demonstrated that AME increased the number of DNA strand breaks in V79 cells treated after 1.5 h in a concentration-dependent manner starting at 0.5 µM, with AME being slightly more effective than AOH at low concentrations (Fleck et al. 2012). In contrast, up to 50 µM AME in KYSE510 (human esophageal carcinoma cell line) and HT29 cells did not significantly increase tail intensity after 1- and 3-h exposure, respectively, in both, the alkaline and

modified version using the fpg enzyme (Tiessen et al. 2013a, 2017). This suggested that oxidative stress did not play a predominant role in the induction of DNA damage. The results are in line with observations demonstrating that, after 24-h exposure, concentrations of up to 25 µM AME could no longer induce DNA strand breaks in HT29 cells, presumably due to intense AME glucuronidation and, thus, detoxification (Pfeiffer et al. 2009b). In contrast, it was recently shown (Tran et al. 2020) that 25 µM AME was able to produce a significant increase in DNA strand breaks in HEK239T cells after 24-h incubation. The only in vivo genotoxicity study on AME showed that 7.35 µg/kg body weight/day orally applied for 28 days induced DNA damage in the blood and liver of male Sprague Dawley rats (Tang et al. 2022).

ATX-II induced a dose-dependent increase in DNA strand breaks from ≥ 0.1 µM in HT29 cells after 1 h of treatment as assessed by the alkaline comet assay. The enzyme fpg significantly enhanced the tail intensity at concentrations ≥ 0.05 µM. At 1 µM, fpg increased the amount of fpg-sensitive sites twofold (Tiessen et al. 2013b). Similarly, the incubation of HT29 and Ishikawa cells with 1 µM ATX-II for 1 h caused DNA damage. The effect was further enhanced in the presence of fpg (Aichinger et al. 2018; Schwarz et al. 2012). Of note, fpg-sensitive sites include not only oxidative damage like 8-oxo-dG but
<table><caption>**Table 4** (continued)</caption><thead><tr><th>Cell line</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>S9 (Y/N)</th><th>Effect</th><th>References</th></tr></thead><tbody><tr><td>HT29</td><td>1</td><td>1</td><td>N</td><td>Standard comet assay; positive<br>Fpg comet assay; positive</td><td>Aichinger et al. (2018)</td></tr><tr><td>HT29</td><td>0.01, 0.05, 0.1, 0.2, 0.5, 1</td><td>1</td><td>N</td><td>Standard comet assay; positive ≥ 0.1 µM<br>Fpg comet assay; positive ≥ 0.05 µM</td><td>Schwarz et al. (2012)</td></tr><tr><td>HT29</td><td>0.01, 0.05, 0.1, 0.2, 0.5, 1</td><td>24</td><td>N</td><td>Standard comet assay; positive ≥ 0.1 µM<br>Fpg comet assay; positive ≥ 0.05 µM</td><td>Schwarz et al. (2012)</td></tr><tr><td>Ishikawa cells</td><td>1</td><td>1</td><td>N</td><td>Standard comet assay; positive<br>Fpg comet assay; positive</td><td>Aichinger et al. (2022a)</td></tr><tr><td colspan="6"><strong>Alkaline unwinding assay</strong></td></tr><tr><td>Caco-2</td><td>0.25, 0.5, 1</td><td>1.5</td><td>N</td><td>≥ 0.25 µM</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>HepG2</td><td>0.25, 0.5, 1</td><td>1.5</td><td>N</td><td>≥ 0.25 µM</td><td>Fleck et al. (2014a, b)</td></tr><tr><td>V79</td><td>0.1, 0.25, 0.5, 1, 5, 10, 20</td><td>1.5</td><td>N</td><td>≥ 0.1 µM</td><td>Fleck et al. (2012)</td></tr><tr><td>V79</td><td>0.25, 0.5, 1</td><td>1.5</td><td>N</td><td>≥ 0.25 µM</td><td>Fleck et al. (2014a, b)</td></tr><tr><td colspan="6"><strong>Tentoxin (TEN)</strong></td></tr><tr><td colspan="6"><strong>Comet assay</strong></td></tr><tr><td>HEK 293 T</td><td>25</td><td>24</td><td>N</td><td>Standard comet assay; negative</td><td>Tran et al. (2020)</td></tr></tbody></table>

Cell lines PNT1A- human primary prostate epithelial cells, HepG2 human liver carcinoma, A431 human vulva carcinoma, Caco-2 human colon carcinoma, HEK 293T human embryonal kidney, HT29 human colorectal adenocarcinoma, KYSE510 human esophageal squamous carcinoma, RAW264.7 mouse macrophage, V79 lung Chinese hamster fibroblasts, Ishikawa endometrial adenocarcinoma cells

AME induced DNA strand breaks in HT29 and A431 cells at concentrations ≥ 10 and ≥ 1 µM, respectively, after 1 h of exposure as assessed in the alkaline comet assay (Fehr et al. 2009), indicating a substantial genotoxic potential. The rate of DNA strand breaks induced by AME did not statistically significantly differ from fpg-treated cells, suggesting that AME does not cause oxidative DNA damage (Fehr et al. 2009). In the alkaline unwinding assay, AME showed slightly but not significantly lower DNA strand-breaking activities as compared to AOH in both cell lines (Pfeiffer et al. 2007). Furthermore, using the same assay, a dose-dependent increase (> 6.25 µM) of DNA strand breaks in HT29, HepG2, and V79 cells after 1 h was revealed (Pfeiffer et al. 2007). At concentrations above 10 µM, AME induced DNA double-strand breaks in A431 cells in the comet assay under neutral pH conditions after 1 h (Fehr et al. 2010). Similarly, using the alkaline unwinding assay, it was demonstrated that AME increased the number of DNA strand breaks in V79 cells treated after 1.5 h in a concentration-dependent manner starting at 0.5 µM, with AME being slightly more effective than AOH at low concentrations (Fleck et al. 2012). In contrast, up to 50 µM AME in KYSE510 (human esophageal carcinoma cell line) and HT29 cells did not significantly increase tail intensity after 1- and 3-h exposure, respectively, in both, the alkaline and

modified version using the fpg enzyme (Tiessen et al. 2013a, 2017). This suggested that oxidative stress did not play a predominant role in the induction of DNA damage. The results are in line with observations demonstrating that, after 24-h exposure, concentrations of up to 25 µM AME could no longer induce DNA strand breaks in HT29 cells, presumably due to intense AME glucuronidation and, thus, detoxification (Pfeiffer et al. 2009b). In contrast, it was recently shown (Tran et al. 2020) that 25 µM AME was able to produce a significant increase in DNA strand breaks in HEK239T cells after 24-h incubation. The only in vivo genotoxicity study on AME showed that 7.35 µg/kg body weight/day orally applied for 28 days induced DNA damage in the blood and liver of male Sprague Dawley rats (Tang et al. 2022).

ATX-II induced a dose-dependent increase in DNA strand breaks from ≥ 0.1 µM in HT29 cells after 1 h of treatment as assessed by the alkaline comet assay. The enzyme fpg significantly enhanced the tail intensity at concentrations ≥ 0.05 µM. At 1 µM, fpg increased the amount of fpg-sensitive sites twofold (Tiessen et al. 2013b). Similarly, the incubation of HT29 and Ishikawa cells with 1 µM ATX-II for 1 h caused DNA damage. The effect was further enhanced in the presence of fpg (Aichinger et al. 2018; Schwarz et al. 2012). Of note, fpg-sensitive sites include not only oxidative damage like 8-oxo-dG but
<table><caption>**Table 5** Summary of published in vivo studies reporting the genotoxicity or mutagenicity of *Alternaria* toxins</caption><thead><tr><th>Species (strain)</th><th>Route of administration (vehicle)</th><th>Dose (mg/kg bw)</th><th>Timepoint (s)</th><th>Effect (read-out)</th><th>OECD (Y/N)</th><th>References</th></tr></thead><tbody><tr><td colspan="7"><strong>Alternariol (AOH)</strong></td></tr><tr><td colspan="7"><strong>Comet assay</strong></td></tr><tr><td>Male NMRI mice, 5-7 weeks old</td><td>Oral, Corn oil</td><td>3 × 2000 mg/kg in corn oil, at 0, 24 and 45 h</td><td>Animal killed 48 h after first application</td><td>Alkaline comet assay; Liver; negative<br>Stomach; not analyzable<br>Gut not analyzable</td><td>In compliance with GLP</td><td>Schuchardt et al. (2014)</td></tr><tr><td>Male Sprague Dawley rats, 5 weeks old</td><td>Oral, Corn oil</td><td>5.51, 11.03 and 22.05 µg/kg bw</td><td>28 consecutive days</td><td>On day 4<br>Peripheral blood; negative<br>Liver; negative</td><td>Yes OECD 407<br>exposure<br>and comet<br>OECD 489</td><td>Miao et al. (2022)</td></tr><tr><td>Male wistar rats (7-9 weeks old)</td><td>Oral, ethanol and sunflower seed oil</td><td>10 mg/kg</td><td>Single-dose treatment</td><td>Parotid gland; positive<br>Time after treatment and assay performance unknown</td><td>N</td><td>Samak et al. (2019)</td></tr><tr><td colspan="7"><strong>Pig-a assay</strong></td></tr><tr><td>Male Sprague Dawley rats, 5 weeks old</td><td>Oral, Corn oil</td><td>5.51, 11.03 and 22.05 µg/kg bw</td><td>28 consecutive days</td><td>After 28 days, histopathological lesions in liver, kidney, spleen in all AOH groups but no genotoxicity<br>On day 42 (28 days treatment, 14 days recovery), in the AOH-HR (high dose/recovery) group, the rate of Pig-a mutant phenotype reticulo-cytes (RETCD59-) significantly increased<br>On day 56, both RETCD59- and the rate of Pig-a mutant phenotype erythrocytes (RBCCD59-) were significantly reduced</td><td>N</td><td>Miao et al. (2022)</td></tr><tr><td>Micronucleus assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats, 5 weeks old</td><td>Oral, Corn oil</td><td>5.51, 11.03 and 22.05 µg/kg bw</td><td>28 consecutive days</td><td>Negative</td><td>YES, No. 474</td><td>Miao et al. (2022)</td></tr><tr><td colspan="7"><strong>Alternariol monomethyl ether (AME)</strong></td></tr><tr><td colspan="7"><strong>Comet assay</strong></td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, corn oil</td><td>1.84, 3.67, or 7.35 µg/kg body weight/day</td><td>28 days</td><td>On day 4; peripheral blood; positive at 7.35 µg/kg body weight/day, Standard comet assay</td><td>YES, OECD 489</td><td>Tang et al. (2022)</td></tr></tbody></table>

<figure><img src="image_8.png" /></figure>
<table><caption>**Table 5** Summary of published in vivo studies reporting the genotoxicity or mutagenicity of *Alternaria* toxins</caption><thead><tr><th>Species (strain)</th><th>Route of administration (vehicle)</th><th>Dose (mg/kg bw)</th><th>Timepoint (s)</th><th>Effect (read-out)</th><th>OECD (Y/N)</th><th>References</th></tr></thead><tbody><tr><td colspan="7"><strong>Alternariol (AOH)</strong></td></tr><tr><td colspan="7"><strong>Comet assay</strong></td></tr><tr><td>Male NMRI mice, 5-7 weeks old</td><td>Oral, Corn oil</td><td>3 × 2000 mg/kg in corn oil, at 0, 24 and 45 h</td><td>Animal killed 48 h after first application</td><td>Alkaline comet assay; Liver; negative<br>Stomach; not analyzable<br>Gut not analyzable</td><td>In compliance with GLP</td><td>Schuchardt et al. (2014)</td></tr><tr><td>Male Sprague Dawley rats, 5 weeks old</td><td>Oral, Corn oil</td><td>5.51, 11.03 and 22.05 µg/ kg bw</td><td>28 consecutive days</td><td>On day 4<br>Peripheral blood; negative<br>Liver; negative</td><td>Yes OECD 407<br>exposure<br>and comet<br>OECD 489</td><td>Miao et al. (2022)</td></tr><tr><td>Male wistar rats (7-9 weeks old)</td><td>Oral, ethanol and sunflower seed oil</td><td>10 mg/kg</td><td>Single-dose treatment</td><td>Parotid gland; positive<br>Time after treatment and assay performance unknown</td><td>N</td><td>Samak et al. (2019)</td></tr><tr><td colspan="7"><strong>Pig-a assay</strong></td></tr><tr><td>Male Sprague Dawley rats, 5 weeks old</td><td>Oral, Corn oil</td><td>5.51, 11.03 and 22.05 µg/ kg bw</td><td>28 consecutive days</td><td>After 28 days, histopathological lesions in liver, kidney, spleen in all AOH groups but no genotoxicity<br>On day 42 (28 days treatment, 14 days recovery), in the AOH-HR (high dose/recovery) group, the rate of Pig-a mutant phenotype reticulo-cytes (RETCD59-) significantly increased<br>On day 56, both RETCD59- and the rate of Pig-a mutant phenotype erythrocytes (RBCCD59-) were significantly reduced</td><td>N</td><td>Miao et al. (2022)</td></tr><tr><td>Micronucleus assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats, 5 weeks old</td><td>Oral, Corn oil</td><td>5.51, 11.03 and 22.05 µg/ kg bw</td><td>28 consecutive days</td><td>Negative</td><td>YES, No. 474</td><td>Miao et al. (2022)</td></tr><tr><td colspan="7"><strong>Alternariol monomethyl ether (AME)</strong></td></tr><tr><td colspan="7"><strong>Comet assay</strong></td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, corn oil</td><td>1.84, 3.67, or 7.35 µg/kg body weight/day</td><td>28 days</td><td>On day 4; peripheral blood; positive at 7.35 µg/ kg body weight/day, Standard comet assay</td><td>YES, OECD 489</td><td>Tang et al. (2022)</td></tr></tbody></table>

<figure><img src="image_8.png" /></figure>
<table><caption>**Table 5 (continued)**</caption><thead><tr><th>Species (strain)</th><th>Route of administration (vehicle)</th><th>Dose (mg/kg bw)</th><th>Timepoint (s)</th><th>Effect (read-out)</th><th>OECD (Y/N)</th><th>References</th></tr></thead><tbody><tr><td>Male Sprague Dawley rats</td><td>Oral, corn oil</td><td>1.84, 3.67, or 7.35 µg/kg body weight/day</td><td>28 days</td><td>On day 28; peripheral blood, positive at 7.35 µg/kg body weight/day<br>Liver; positive at 7.35 µg/kg body weight/day<br>Standard comet assay</td><td>YES, OECD 489</td><td>Tang et al. (2022)</td></tr><tr><td>Pig-a assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats treated daily for 28 days with oral gavage</td><td>Oral, corn oil</td><td>1.84, 3.67, or 7.35 µg/kg body weight/day</td><td>28 days</td><td>Increase in RETCD59 – in highest AME group. After the 14-day recovery period, RETCD59 – was Significantly decreased compared to the value on day 28 (P&lt;0.05)</td><td>N</td><td>Tang et al. (2022)</td></tr><tr><td>Micronucleus assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats treated daily for 28 days with oral gavage</td><td>Oral, corn oil</td><td>1.84, 3.67, or 7.35 µg/kg body weight/day</td><td>28 days</td><td>On day 28; Bone narrow; positive at 7.35 µg/kg body weight/day AME<br>Peripheral blood; positive at AME 7.35 µg/kg body weight/day, but decrease of MN after 14 days of recovery</td><td>N</td><td>Tang et al. (2022)</td></tr><tr><td>Tenuazonic acid (TeA) no studies available</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Altertoxin I (ATX-I) Comet assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, Corn oil,</td><td>1.10 and 5.51 µg/kg bw/d</td><td>28 days</td><td>Day 4; peripheral blood; negative Standard comet assay</td><td>Yes, OECD 489</td><td>Zhu et al. (2022)</td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, Corn oil,</td><td>1.10 and 5.51 µg/kg bw/d</td><td>28 days</td><td>Day 28; peripheral blood; Positive at 5.51 µg/kg bw/d<br>Standard comet assay</td><td>Yes, OECD 489</td><td>Zhu et al. (2022)</td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, Corn oil,</td><td>1.10 and 5.51 µg/kg bw/d</td><td>28 days</td><td>Day 29; liver; Positive at 5.51 µg/kg bw/d<br>Standard comet assay</td><td>Yes, OECD 489</td><td>Zhu et al. (2022)</td></tr><tr><td>Pig-a assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, Corn oil,</td><td>1.10 and 5.51 µg/kg bw/d</td><td>28 days</td><td>Samples collected on Days 0, 14, and 28<br>No increase in the frequencies of mutant RBC and RET were observed on Days 14 and 28, indicating that ATX-I did not induce erythrocytes mutation in the testing dose range</td><td>N</td><td>Zhu et al. (2022)</td></tr></tbody></table>

<figure><img src="image_9.png" /></figure>
<table><caption>**Table 5 (continued)**</caption><thead><tr><th>Species (strain)</th><th>Route of administration (vehicle)</th><th>Dose (mg/kg bw)</th><th>Timepoint (s)</th><th>Effect (read-out)</th><th>OECD (Y/N)</th><th>References</th></tr></thead><tbody><tr><td>Male Sprague Dawley rats</td><td>Oral, corn oil</td><td>1.84, 3.67, or 7.35 µg/kg body weight/day</td><td>28 days</td><td>On day 28; <u>peripheral blood</u>, positive at 7.35 µg/kg body weight/day<br><u>Liver</u>; positive at 7.35 µg/kg body weight/day<br>Standard comet assay</td><td>YES, OECD 489</td><td>Tang et al. (2022)</td></tr><tr><td>Pig-a assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats treated daily for 28 days with oral gavage</td><td>Oral, corn oil</td><td>1.84, 3.67, or 7.35 µg/kg body weight/day</td><td>28 days</td><td>Increase in RETCD59 – in highest AME group. After the 14-day recovery period, RETCD59 – was Significantly decreased compared to the value on day 28 (P&lt;0.05)</td><td>N</td><td>Tang et al. (2022)</td></tr><tr><td>Micronucleus assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats treated daily for 28 days with oral gavage</td><td>Oral, corn oil</td><td>1.84, 3.67, or 7.35 µg/kg body weight/day</td><td>28 days</td><td>On day 28; <u>Bone marrow</u>; positive at 7.35 µg/kg body weight/day<br><u>Peripheral blood</u>; positive at AME 7.35 µg/kg body weight/day, but decrease of MN after 14 days of recovery</td><td>N</td><td>Tang et al. (2022)</td></tr><tr><td>Tenuazonic acid (TeA) no studies available</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Altertoxin I (ATX-I)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Comet assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, Corn oil,</td><td>1.10 and 5.51 µg/kg bw/d</td><td>28 days</td><td>Day 4; <u>peripheral blood</u>; negative Standard comet assay</td><td>Yes, OECD 489</td><td>Zhu et al. (2022)</td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, Corn oil,</td><td>1.10 and 5.51 µg/kg bw/d</td><td>28 days</td><td>Day 28; <u>peripheral blood</u>; Positive at 5.51 µg/kg bw/d<br>Standard comet assay</td><td>Yes, OECD 489</td><td>Zhu et al. (2022)</td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, Corn oil,</td><td>1.10 and 5.51 µg/kg bw/d</td><td>28 days</td><td>Day 29; <u>liver</u>; Positive at 5.51 µg/kg bw/d<br>Standard comet assay</td><td>Yes, OECD 489</td><td>Zhu et al. (2022)</td></tr><tr><td>Pig-a assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, Corn oil,</td><td>1.10 and 5.51 µg/kg bw/d</td><td>28 days</td><td>Samples collected on Days 0, 14, and 28<br>No increase in the frequencies of mutant RBC and RET were observed on Days 14 and 28, indicating that ATX-I did not induce erythrocytes mutation in the testing dose range</td><td>N</td><td>Zhu et al. (2022)</td></tr></tbody></table>

<figure><img src="image_9.png" /></figure>
<table><caption>**Table 5 (continued)**</caption><thead><tr><th>Species (strain)</th><th>Route of administration (vehicle)</th><th>Dose (mg/kg bw)</th><th>Timepoint (s)</th><th>Effect (read-out)</th><th>OECD (Y/N)</th><th>References</th></tr></thead><tbody><tr><td>Micronucleus assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, Corn oil,</td><td>1.10 and 5.51 µg/kg bw/d</td><td>28 days</td><td>Blood collected on days 0, 4, 14, and 28; MN frequencies in peripheral blood and bone marrow did not differ among groups at any time point</td><td>Y (TG 474)</td><td>Zhu et al. (2022)</td></tr><tr><td>Altertoxin II (ATX-II)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>γH2AX in the colon</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, 1:10 (v/v) mixture of ethanol and sunflower seed oil</td><td>0.21 mg/kg bw</td><td>Single-dose treatment</td><td>Killing after 3 h or 24 h, respectively. After 24 h, enhanced levels of γH2AX in the colon</td><td>N</td><td>Aichinger et al. (2022b)</td></tr><tr><td>Altertoxin III (ATX-III) no studies available</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Altenuene (ALT) no studies available</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

also other modifications like ring-open formamidopyrimidine structures which might arise, e.g., from N7-guanine adduct formation. ATX-II induced DNA strand breaks in HT29 cells even after prolonged exposure for 24 h, with a similar effect as a 1-h exposure and independent of the presence of fpg (Schwarz et al. 2012). Furthermore, the co-treatment of Ishikawa and HT29 cells with antioxidants (delphinidin and N-acetyl cysteine) and ATX-II (1 µM) significantly reduced the mycotoxin-induced genotoxicity, or even completely suppressed DNA damage, both with and without fpg treatment (Aichinger et al. 2018, 2020c). In contrast, DNA breaking properties in HT29 cells were not reduced, when the cells were exposed for 1 h to 1 µM ATX-II after 24-h pre-incubation with delphinidin, as measured in the comet assay (Aichinger et al. 2018). This demonstrated that pre-incubation with the polyphenol is insufficient and that only co-exposure leads to a suppression of genotoxicity.

ATX-II caused also DNA strand breaks in V79, HepG2 and Caco-2 cells treated for 1.5 h (≥ 0.25 µM), with no statistically significant differences between the cell lines (Fleck et al. 2012; Fleck et al. 2014a, b). It was, thus, concluded that ATX-II has a significantly higher genotoxic potential than AME or AOH, because it could induce DNA strand breaks at lower concentrations and with greater effect than the other two toxins when tested in the same cell lines (Fleck et al. 2012; Fleck et al. 2014a, b; Tiessen et al. 2013a). The high potency of ATX-II results likely from the epoxide group in the molecule that can react with DNA without metabolic activation in contrast to AME and AOH (Fleck et al. 2012; Soukup et al. 2020). So far, ATX-II was only investigated in one in vivo study in rats with a single bolus application, resulting in enhanced levels of γH2AX in the colon of the animals after 24 h (Aichinger et al. 2022b; Puntscher et al. 2019a).

Little is known about the genotoxicity of ATX-I. In V79, HepG2, and Caco-2 cells, ATX-I induced DNA strand breaks after 1.5 h as determined in the alkaline unwinding assay, with no differences between the cell lines (Fleck et al. 2014a, b). Compared to ATX-II, ATX-I was less potent but had still considerable DNA strand-breaking potency with about the same genotoxic activity as AOH (Fleck et al. 2014a, b). Recently, ATX-I (5.51 µg/kg bw/d) was reported to induce DNA strand breaks in the peripheral blood and liver of male Sprague Dawley rats exposed orally for 28 days (28-day repeated administration in vivo) (Zhu et al. 2022).

There is to our knowledge only one study available investigating the genotoxic activity of ALT. Using the comet assay, it showed that up to 100 µM ALT did not induce DNA strand breaks in A431 and HT29 cells after 1-h incubation (Fehr et al. 2009). Comparably, there is only one published genotoxicity study on TeA, demonstrating that up to 200 µM toxin did not induce DNA strand breaks in HT29 cells after

<figure><img src="image_10.png" /></figure>
<table><caption>**Table 5 (continued)**</caption><thead><tr><th>Species (strain)</th><th>Route of administration (vehicle)</th><th>Dose (mg/kg bw)</th><th>Timepoint (s)</th><th>Effect (read-out)</th><th>OECD (Y/N)</th><th>References</th></tr></thead><tbody><tr><td>Micronucleus assay</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, Corn oil,</td><td>1.10 and 5.51 µg/kg bw/d</td><td>28 days</td><td>Blood collected on days 0, 4, 14, and 28; MN frequencies in peripheral blood and bone marrow did not differ among groups at any time point</td><td>Y (TG 474)</td><td>Zhu et al. (2022)</td></tr><tr><td>Altertoxin II (ATX-II)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>γH2AX in the colon</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male Sprague Dawley rats</td><td>Oral, 1:10 (v/v) mixture of ethanol and sunflower seed oil</td><td>0.21 mg/kg bw</td><td>Single-dose treatment</td><td>Killing after 3 h or 24 h, respectively. After 24 h, enhanced levels of γH2AX in the colon</td><td>N</td><td>Aichinger et al. (2022b)</td></tr><tr><td>Altertoxin III (ATX-III) no studies available</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Altenuene (ALT) no studies available</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

also other modifications like ring-open formamidopyrimidine structures which might arise, e.g., from N7-guanine adduct formation. ATX-II induced DNA strand breaks in HT29 cells even after prolonged exposure for 24 h, with a similar effect as a 1-h exposure and independent of the presence of fpg (Schwarz et al. 2012). Furthermore, the co-treatment of Ishikawa and HT29 cells with antioxidants (delphinidin and N-acetyl cysteine) and ATX-II (1 µM) significantly reduced the mycotoxin-induced genotoxicity, or even completely suppressed DNA damage, both with and without fpg treatment (Aichinger et al. 2018, 2020c). In contrast, DNA breaking properties in HT29 cells were not reduced, when the cells were exposed for 1 h to 1 µM ATX-II after 24-h pre-incubation with delphinidin, as measured in the comet assay (Aichinger et al. 2018). This demonstrated that pre-incubation with the polyphenol is insufficient and that only co-exposure leads to a suppression of genotoxicity.

ATX-II caused also DNA strand breaks in V79, HepG2 and Caco-2 cells treated for 1.5 h (≥ 0.25 µM), with no statistically significant differences between the cell lines (Fleck et al. 2012; Fleck et al. 2014a, b). It was, thus, concluded that ATX-II has a significantly higher genotoxic potential than AME or AOH, because it could induce DNA strand breaks at lower concentrations and with greater effect than the other two toxins when tested in the same cell lines (Fleck et al. 2012; Fleck et al. 2014a, b; Tiessen et al. 2013a). The high potency of ATX-II results likely from the epoxide group in the molecule that can react with DNA without metabolic activation in contrast to AME and AOH (Fleck et al. 2012; Soukup et al. 2020). So far, ATX-II was only investigated in one in vivo study in rats with a single bolus application, resulting in enhanced levels of γH2AX in the colon of the animals after 24 h (Aichinger et al. 2022b; Puntscher et al. 2019a).

Little is known about the genotoxicity of ATX-I. In V79, HepG2, and Caco-2 cells, ATX-I induced DNA strand breaks after 1.5 h as determined in the alkaline unwinding assay, with no differences between the cell lines (Fleck et al. 2014a, b). Compared to ATX-II, ATX-I was less potent but had still considerable DNA strand-breaking potency with about the same genotoxic activity as AOH (Fleck et al. 2014a, b). Recently, ATX-I (5.51 µg/kg bw/d) was reported to induce DNA strand breaks in the peripheral blood and liver of male Sprague Dawley rats exposed orally for 28 days (28-day repeated administration in vivo) (Zhu et al. 2022).

There is to our knowledge only one study available investigating the genotoxic activity of ALT. Using the comet assay, it showed that up to 100 µM ALT did not induce DNA strand breaks in A431 and HT29 cells after 1-h incubation (Fehr et al. 2009). Comparably, there is only one published genotoxicity study on TeA, demonstrating that up to 200 µM toxin did not induce DNA strand breaks in HT29 cells after

<figure><img src="image_10.png" /></figure>
1 h, using the standard alkaline comet assay and a modified version with fpg enzyme (Schwarz et al. 2012). The only available study on the genotoxicity of TEN showed that TEN did not induce statistically significant DNA strand breaks in HEK293T cells treated with 25 µM for 24 h (Tran et al. 2020).

Under cell-free conditions, AOH interfered with human topoisomerases I, IIα and IIβ, with a preference for the IIα-isof orm (Fehr et al. 2009). In cell culture (A431 cells), sta- bilization of the covalent DNA-topoisomerase intermediate was observed at DNA-damaging concentrations, confirm- ing AOH as a topoisomerase poison (Fehr et al. 2009). The connection between targeting this enzyme and the toxin's genotoxic effectivity was further apparent by its impact on human tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme vital for the repair of trapped DNA-topoisomerase intermediates (Fehr et al. 2010). Of note, the targeting of human topoisomerases might mechanistically be associated with the activity of AOH (and other *Alternaria* toxins) on bacterial gyrase (Jarolim et al. 2017), which is presumably vital in the defence of the mould's ecological nice against other microorganisms. In addition to AOH, several other *Alternaria* toxins were found to target human topoisom- erases as well as bacterial gyrase. Under cell-free conditions (decatenation assay), the potency to inhibit human topo- isomerase II activity declined in the order STTX-III (initial inhibitory concentration 10 µM) > AOH (25 µM) = AME (25 µM) = ALS (25 µM) = ATX-II (25 µM) > ALN (50 µM) = ATX-I (50 µM) > ALP (75 µM). Inhibition of gyrase activity was most pronounced for AOH and AME (initial inhibitory concentration 10 µM) followed by ATX-II (25 µM) > ATX-I = ALP = STTX-III (50 µM) (Jarolim et al. 2017; Tiessen et al. 2013b). In contrast to AOH, the DNA- damaging potential of ATX-II was already observed clearly at concentrations below those affecting topoisomerase activ- ity, thus giving rise to the hypothesis of DNA adduct forma- tion by the epoxide-bearing perylene quinone (Fleck et al. 2016). Indeed, under cell-free conditions, ATX-II was found to form covalent guanine adducts (Soukup et al. 2020). Stud- ies on the formation of DNA adducts in cell culture or under in vivo conditions are not available so far.

TEN and TeA did not produce DNA double-strand breaks (DSB) in liver cells as shown in the H2AX assay (Hessel-Pras et al. 2019), while AOH and AME increased DSB in several cell types (Kowalska et al. 2021a; Solhaug et al. 2012, 2015).

Only a few studies have investigated the potential of *Alternaria* toxins to induce chromosome damage. AOH increased micronucleus (MN) formation in both Ishikawa and V79 cells (Lehmann et al. 2006). In Ishikawa cells, the number of MN-containing cells was significantly increased after AOH treatment with 5 and 10 µM. At 10 µM, there was a statistically significant increase in the number of

kinetochore-negative MN as visualized by fluorescence-labeled antikinetochore antibodies. This indicated that MN formation resulted from a clastogenic action of AOH. Further studies on the genotoxic potential of AOH were performed in Chinese hamster V79 cells as a relatively high frequency of distorted nuclei and multipolar or misaligned mitotic spindles was present in the AOH-treated Ishikawa cells. The V79 cells were treated for 6 h with AOH at concentrations ranging from 2.5 µM to 50 µM and either analyzed directly after the treatment, or after an AOH-free incubation period of 3 h, 14 h, or 24 h. A clear concentration-dependent increase in the MN frequency was detected after treatment with 5–50 µM AOH for 6 h followed by post-incubation for 14 h or 24 h. Like in Ishikawa cells, MN induced by AOH treatment predominantly contained chromosome fragments (Lehmann et al. 2006). AOH exposure (30 µM, 24 h) also resulted in an increased incidence of micronuclei in RAW264.7 mouse macrophages (Solhaug et al. 2013). All in vitro experiments showing the formation of micronuclei in the presence of AOH were performed in the absence of an external metabolic system.

The induction of MN formation in vitro by AOH is in line with the observation of Brugger et al. (2006) that AOH predominantly increased the formation of small colonies in the thymidine kinase (TK) assay, which are indicative of extensive chromosomal deletions. However, the positive in vitro MN results with AOH were not confirmed in vivo. In a recent multi-endpoint study of Miao et al. male Sprague Dawley rats received a low, medium, or high dose (i.e., 5.51, 10.03, or 22.05 µg/kg bw/day) of AOH by oral gavage for 28 consecutive days, with and without a recovery period of 14 days (Miao et al. 2022). No increase in MN frequency in the reticulocytes collected from the peripheral blood and the bone marrow was observed for any of the AOH exposure scenarios (Miao et al. 2022). According to the authors, the discrepancy between the in vitro and in vivo MN results might relate to the toxicokinetic properties of AOH. No toxicity was observed in the bone marrow; thus, the question arises whether a toxicologically relevant proportion of AOH might reach the bone marrow. A previous study on the kinetics of AOH had indeed revealed that the absorption rate from the gastrointestinal tract was very low, although it should be highlighted that this study was done in NMRI mice and not in rats (Schuchardt et al. 2014). Alternatively, the authors considered that the in vivo MN test might not be the most sensitive assay to detect AOH-induced genotoxic effects.

Similar multi-endpoint studies were performed with AME and ATX-I. For ATX-I, no effect on the in vivo MN formation was observed in male Sprague Dawley rats after treatment with either 1.10 or 5.51 µg/kg bw/day for 28 consecutive days via oral gavage (Zhu et al. 2022). In contrast, the in vivo MN study with AME revealed a dose-dependent increase in MN frequency in both peripheral blood and bone
1 h, using the standard alkaline comet assay and a modified version with fpg enzyme (Schwarz et al. 2012). The only available study on the genotoxicity of TEN showed that TEN did not induce statistically significant DNA strand breaks in HEK293T cells treated with 25 µM for 24 h (Tran et al. 2020).

Under cell-free conditions, AOH interfered with human topoisomerases I, IIα and IIβ, with a preference for the IIα-isoeform (Fehr et al. 2009). In cell culture (A431 cells), stabilization of the covalent DNA-topoisomerase intermediate was observed at DNA-damaging concentrations, confirming AOH as a topoisomerase poison (Fehr et al. 2009). The connection between targeting this enzyme and the toxin's genotoxic effectivity was further apparent by its impact on human tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme vital for the repair of trapped DNA-topoisomerase intermediates (Fehr et al. 2010). Of note, the targeting of human topoisomerases might mechanistically be associated with the activity of AOH (and other *Alternaria* toxins) on bacterial gyrase (Jarolim et al. 2017), which is presumably vital in the defence of the mould's ecological nice against other microorganisms. In addition to AOH, several other *Alternaria* toxins were found to target human topoisomerases as well as bacterial gyrase. Under cell-free conditions (decatenation assay), the potency to inhibit human topoisomerase II activity declined in the order STTX-III (initial inhibitory concentration 10 µM) > AOH (25 µM) = AME (25 µM) = ALS (25 µM) = ATX-II (25 µM) > ALN (50 µM) = ATX-I (50 µM) > ALP (75 µM). Inhibition of gyrase activity was most pronounced for AOH and AME (initial inhibitory concentration 10 µM) followed by ATX-II (25 µM) > ATX-I = ALP = STTX-III (50 µM) (Jarolim et al. 2017; Tiessen et al. 2013b). In contrast to AOH, the DNA-damaging potential of ATX-II was already observed clearly at concentrations below those affecting topoisomerase activity, thus giving rise to the hypothesis of DNA adduct formation by the epoxide-bearing perylene quinone (Fleck et al. 2016). Indeed, under cell-free conditions, ATX-II was found to form covalent guanine adducts (Soukup et al. 2020). Studies on the formation of DNA adducts in cell culture or under in vivo conditions are not available so far.

TEN and TeA did not produce DNA double-strand breaks (DSB) in liver cells as shown in the H2AX assay (Hessel-Pras et al. 2019), while AOH and AME increased DSB in several cell types (Kowalska et al. 2021a; Solhaug et al. 2012, 2015).

Only a few studies have investigated the potential of *Alternaria* toxins to induce chromosome damage. AOH increased micronucleus (MN) formation in both Ishikawa and V79 cells (Lehmann et al. 2006). In Ishikawa cells, the number of MN-containing cells was significantly increased after AOH treatment with 5 and 10 µM. At 10 µM, there was a statistically significant increase in the number of

kinetochore-negative MN as visualized by fluorescence-labeled antikinetochore antibodies. This indicated that MN formation resulted from a clastogenic action of AOH. Further studies on the genotoxic potential of AOH were performed in Chinese hamster V79 cells as a relatively high frequency of distorted nuclei and multipolar or misaligned mitotic spindles was present in the AOH-treated Ishikawa cells. The V79 cells were treated for 6 h with AOH at concentrations ranging from 2.5 µM to 50 µM and either analyzed directly after the treatment, or after an AOH-free incubation period of 3 h, 14 h, or 24 h. A clear concentration-dependent increase in the MN frequency was detected after treatment with 5–50 µM AOH for 6 h followed by post-incubation for 14 h or 24 h. Like in Ishikawa cells, MN induced by AOH treatment predominantly contained chromosome fragments (Lehmann et al. 2006). AOH exposure (30 µM, 24 h) also resulted in an increased incidence of micronuclei in RAW264.7 mouse macrophages (Solhaug et al. 2013). All in vitro experiments showing the formation of micronuclei in the presence of AOH were performed in the absence of an external metabolic system.

The induction of MN formation in vitro by AOH is in line with the observation of Brugger et al. (2006) that AOH predominantly increased the formation of small colonies in the thymidine kinase (TK) assay, which are indicative of extensive chromosomal deletions. However, the positive in vitro MN results with AOH were not confirmed in vivo. In a recent multi-endpoint study of Miao et al. male Sprague Dawley rats received a low, medium, or high dose (i.e., 5.51, 10.03, or 22.05 µg/kg bw/day) of AOH by oral gavage for 28 consecutive days, with and without a recovery period of 14 days (Miao et al. 2022). No increase in MN frequency in the reticulocytes collected from the peripheral blood and the bone marrow was observed for any of the AOH exposure scenarios (Miao et al. 2022). According to the authors, the discrepancy between the in vitro and in vivo MN results might relate to the toxicokinetic properties of AOH. No toxicity was observed in the bone marrow; thus, the question arises whether a toxicologically relevant proportion of AOH might reach the bone marrow. A previous study on the kinetics of AOH had indeed revealed that the absorption rate from the gastrointestinal tract was very low, although it should be highlighted that this study was done in NMRI mice and not in rats (Schuchardt et al. 2014). Alternatively, the authors considered that the in vivo MN test might not be the most sensitive assay to detect AOH-induced genotoxic effects.

Similar multi-endpoint studies were performed with AME and ATX-I. For ATX-I, no effect on the in vivo MN formation was observed in male Sprague Dawley rats after treatment with either 1.10 or 5.51 µg/kg bw/day for 28 consecutive days via oral gavage (Zhu et al. 2022). In contrast, the in vivo MN study with AME revealed a dose-dependent increase in MN frequency in both peripheral blood and bone